Compositions of aminoacyl-tRNA synthetase and uses thereof

ABSTRACT

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNA&#39;s, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNA&#39;s/synthetases are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins using these orthogonal pairs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/292,903 filed on Dec. 1, 2005, and claims the benefit of priority toU.S. provisional patent application Ser. No. 60/639,146, filed Dec. 22,2004, the specification and disclosure of which is incorporated hereinin its entirety for all purposes.

FIELD OF THE PRESENT INVENTION

The invention pertains to the field of translation biochemistry. Theinvention relates to methods for producing and compositions ofaminoacyl-tRNA synthetases and uses thereof. The invention also relatesto methods of producing proteins in cells using such aminoacyl-tRNAsynthetases and related compositions.

BACKGROUND OF THE PRESENT INVENTION

The genetic code of every known organism, from bacteria to humans,encodes the same twenty common amino acids. Different combinations ofthe same twenty natural amino acids form proteins that carry outvirtually all the complex processes of life, from photosynthesis tosignal transduction and the immune response. In order to study andmodify protein structure and function, scientists have attempted tomanipulate both the genetic code and the amino acid sequence of protein.However, it has been difficult to remove the constraints imposed by thegenetic code that limit proteins to twenty genetically encoded standardbuilding blocks (with the rare exception of selenocysteine (see, e.g.,A. Bock et al., (1991), Molecular Microbiology 5:515-20) and pyrrolysine(see, e.g., G. Srinivasan, et al., (2002), Science 296:1459-62).

Some progress has been made to remove these constraints, although thisprogress has been limited and the ability to rationally control proteinstructure and function is still in its infancy. For example, chemistshave developed methods and strategies to synthesize and manipulate thestructures of small molecules (see, e.g., E. J. Corey, & X.-M. Cheng,The Logic of Chemical Synthesis (Wiley-Interscience, New York, 1995)).Total synthesis (see, e.g., B. Merrifield, (1986), Science 232:341-7(1986)), and semi-synthetic methodologies (see, e.g., D. Y. Jackson etal., (1994) Science 266:243-7; and, P. E. Dawson, & S. B. Kent, (2000),Annual Review of Biochemistry 69:923-60), have made it possible tosynthesize peptides and small proteins, but these methodologies havelimited utility with proteins over 10 kilo Daltons (kDa). Mutagenesismethods, though powerful, are restricted to a limited number ofstructural changes. In a number of cases, it has been possible tocompetitively incorporate close structural analogues of common aminoacids throughout proteins. See, e.g., R. Furter, (1998), Protein Science7:419-26; K. Kirshenbaum, et al., (2002), Chem Bio Chem 3:235-7; and, V.Doring et al., (2001), Science 292:501-4. Chemical peptide ligation andnative chemical ligation are described in U.S. Pat. No. 6,184,344, U.S.Patent Publication No. 2004/0138412, U.S. Patent Publication No.2003/0208046, WO 02/098902, and WO 03/042235, which are incorporated byreference herein. Lu et al. in Mol Cell. 2001 October; 8(4):759-69describe a method in which a protein is chemically ligated to asynthetic peptide containing unnatural amino acids (expressed proteinligation).

Early work demonstrated that the translational machinery of E. coliwould accommodate amino acids similar in structure to the common twenty.See, Hortin, G., and Boime, I. (1983) Methods Enzymol. 96:777-784. Thiswork was further extended by relaxing the specificity of endogenous E.coli synthetases so that they activate unnatural amino acids as well astheir cognate natural amino acid. Moreover, it was shown that mutationsin editing domains could also be used to extend the substrate scope ofthe endogenous synthetase. See, Doring, V., et al., (2001) Science292:501-504. However, these strategies are limited to recoding thegenetic code rather than expanding the genetic code and lead to varyingdegrees of substitution of one of the common twenty amino acids with anunnatural amino acid.

Later it was shown that unnatural amino acids could be site-specificallyincorporated into proteins in vitro by the addition of chemicallyaminoacylated orthogonal amber suppressor tRNA's to an in vitrotranscription/translation reaction. See, e.g., Noren, C. J., et al.(1989) Science 244:182-188; Bain, J. D., et al., (1989) J. Am. Chem.Soc. 111:8013-8014; Dougherty, D. A. (2000) Curr. Opin. Chem. Biol. 4,645-652; Cornish, V. W., et al. (1995) Angew. Chem., Int. Ed.34:621-633; J. A. Ellman, et al., (1992), Science 255:197-200; and, D.Mendel, et al., (1995), Annual Review of Biophysics and BiomolecularStructure 24:435-462. These studies show that the ribosome andtranslation factors are compatible with a large number of unnaturalamino acids, even those with unusual structures. Unfortunately, thechemical aminoacylation of tRNA's is difficult, and the stoichiometricnature of this process severely limited the amount of protein that couldbe generated.

Unnatural amino acids have been microinjected into cells. For example,unnatural amino acids were introduced into the nicotinic acetylcholinereceptor in Xenopus oocytes (e.g., M. W. Nowak, et al. (1998), In vivoincorporation of unnatural amino acids into ion channels in Xenopusoocyte expression system, Method Enzymol. 293:504-529) by microinjectionof a chemically misacylated Tetrahymena thermophila tRNA (e.g., M. E.Saks, et al. (1996), An engineered Tetrahymena tRNAGln for in vivoincorporation of unnatural amino acids into proteins by nonsensesuppression, J. Biol. Chem. 271:23169-23175), and the relevant mRNA.See, also, D. A. Dougherty (2000), Unnatural amino acids as probes ofprotein structure and function, Curr. Opin. Chem. Biol. 4:645-652 and M.W. Nowak, P. C. Kearney, J. R. Sampson, M. E. Saks, C. G. Labarca, S. K.Silverman, W. G. Zhong, J. Thorson, J. N. Abelson, N. Davidson, P. G.Schultz, D. A. Dougherty and H. A. Lester, Science, 268:439 (1995). AXenopus oocyte was coinjected with two RNA species made in vitro: anmRNA encoding the target protein with a UAG stop codon at the amino acidposition of interest and an amber suppressor tRNA aminoacylated with thedesired unnatural amino acid. The translational machinery of the oocytethen inserts the unnatural amino acid at the position specified by UAG.Unfortunately, this methodology is limited to proteins in cells that canbe microinjected, and because the relevant tRNA is chemically acylatedin vitro, and cannot be re-acylated, the yields of protein are very low.

To overcome these limitations, new components, e.g., orthogonal tRNA's,orthogonal aminoacyl-tRNA synthetases and pairs thereof, were added tothe protein biosynthetic machinery of the prokaryote Escherichia coli(E. coli) (see e.g., L. Wang, et al., (2001), Science 292:498-500) andthe eukaryote Sacchromyces cerevisiae (S. cerevisiae) (e.g., J. Chin etal., Science 301:964-7 (2003)) which has enabled the incorporation ofnon-genetically encoded amino acids to proteins in vivo. A number of newamino acids with novel chemical, physical or biological properties,including photoaffinity labels and photoisomerizable amino acids,photocrosslinking amino acids (see, e.g., Chin, J. W., et al. (2002)Proc. Natl. Acad. Sci. U.S.A. 99:11020-11024; and, Chin, J. W., et al.,(2002) J. Am. Chem. Soc. 124:9026-9027), keto amino acids (see, e.g.,Wang, L., et al., (2003) Proc. Natl. Acad. Sci. U.S.A. 100:56-61 andZhang, Z. et al., Biochem. 42(22):6735-6746 (2003)), heavy atomcontaining amino acids, and glycosylated amino acids have beenincorporated efficiently and with high fidelity into proteins in E. coliand in yeast in response to, e.g., the amber codon (TAG), using thismethodology. See, e.g., J. W. Chin, & P. G. Schultz, (2002), Chem BioChem 3(11):1135-1137 and, L. Wang, & P. G. Schultz, (2002), Chem. Comm.1:1-11.

Several other orthogonal pairs have been reported. Glutaminyl (see,e.g., Liu, D. R., and Schultz, P. G. (1999) Proc. Natl. Acad. Sci.U.S.A. 96:4780-4785), aspartyl (see, e.g., Pastrnak, M., et al., (2000)Helv. Chim. Acta 83:2277-2286), and tyrosyl (see, e.g., Ohno, S., etal., (1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and, Kowal, A. K.,et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) systemsderived from S. cerevisiae tRNA's and synthetases have been describedfor the potential incorporation of unnatural amino acids in E. coli.Systems derived from the E. coli glutaminyl (see, e.g., Kowal, A. K., etal., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) and tyrosyl(see, e.g., Edwards, H., and Schimmel, P. (1990) Mol. Cell. Biol.10:1633-1641) synthetases have been described for use in S. cerevisiae.The E. coli tyrosyl system has been used for the incorporation of3-iodo-L-tyrosine in vivo, in mammalian cells. See, Sakamoto, K., etal., (2002) Nucleic Acids Res. 30:4692-4699. Typically, these systemshave made use of the amber stop codon. To further expand the geneticcode, there is a need to develop improved and/or additional componentsof the biosynthetic machinery, e.g., aminoacyl-tRNA synthetases. Thisinvention fulfills these and other needs, as will be apparent uponreview of the following disclosure.

SUMMARY OF THE PRESENT INVENTION

To expand the genetic code, the invention provides compositions of andmethods of producing orthogonal aminoacyl-tRNA synthetases.Aminoacyl-tRNA synthetases of the present invention aminoacylate tRNAwith a non-naturally encoded amino acid. These translational componentscan be used to incorporate a selected amino acid in a specific positionin a growing polypeptide chain (during nucleic acid translation) inresponse to a selector codon that is recognized by the tRNA.

Methods of producing a protein in a cell with a selected amino acid at aspecified position are also a feature of the present invention. Forexample, a method includes growing, in an appropriate medium, a cell,where the cell comprises a nucleic acid that comprises at least oneselector codon and encodes a protein; and, providing the selected aminoacid. The cell further comprises: an orthogonal tRNA (O-tRNA) thatfunctions in the cell and recognizes the selector codon; and, anorthogonal aminoacyl-tRNA synthetase (O-RS) that preferentiallyaminoacylates the O-tRNA with the selected amino acid. Typically, theO-tRNA comprises suppression activity in the presence of a cognatesynthetase. A protein produced by this method is also a feature of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—The cloverleaf structure of J17 tRNA with TΨC stem mutation sitesis shown.

FIG. 2—Supression of an amber mutation in human growth hormone is shownusing J17 or J17 mutants (F12, F13, F14) and E9 RS. Total cell lysatefor each sample was analyzed by SDS PAGE.

FIG. 3—Supression of an amber mutation in human growth hormone is shownin different cell lines using F13 and E9 RS.

DEFINITIONS

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to limit the scope of the present invention,which will be limited only by the appended claims. As used herein and inthe appended claims, the singular forms “a”, “an” and “the” includeplural referents unless the content clearly dictates otherwise. Thus,for example, reference to “a cell” includes a combination of two or morecells and includes equivalents thereof known to those of ordinary skillin the art, and so forth. Reference to “bacteria” includes mixtures ofbacteria, and the like.

Unless defined herein and below in the reminder of the specification,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which theinvention pertains.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

Homologous: Proteins and/or protein sequences are “homologous” when theyare derived, naturally or artificially, from a common ancestral proteinor protein sequence. Similarly, nucleic acids and/or nucleic acidsequences are homologous when they are derived, naturally orartificially, from a common ancestral nucleic acid or nucleic acidsequence. For example, any naturally occurring nucleic acid can bemodified by any available mutagenesis method to include one or moreselector codon. When expressed, this mutagenized nucleic acid encodes apolypeptide comprising one or more selected amino acid, e.g. unnaturalamino acid. The mutation process can, of course, additionally alter oneor more standard codon, thereby changing one or more standard amino acidin the resulting mutant protein as well. The one or more standard aminoacid may be changed to an unnatural amino acid or a natural amino acid.Homology is generally inferred from sequence similarity between two ormore nucleic acids or proteins (or sequences thereof). The precisepercentage of similarity between sequences that is useful inestablishing homology varies with the nucleic acid and protein at issue,but as little as 25% sequence similarity is routinely used to establishhomology. Higher levels of sequence similarity, e.g., 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% or more, can also be used to establishhomology. Methods for determining sequence similarity percentages (e.g.,BLASTP and BLASTN using default parameters) are described herein and aregenerally available.

Orthogonal: As used herein, the term “orthogonal” refers to a molecule(e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl tRNAsynthetase (O-RS)) that is used with reduced efficiency by a system ofinterest (e.g., a translational system, e.g., a cell). Orthogonal refersto the inability or reduced efficiency, e.g., less than 20% efficient,less than 10% efficient, less than 5% efficient, or e.g., less than 1%efficient, of an orthogonal tRNA and/or orthogonal RS to function in thetranslation system of interest. For example, an orthogonal tRNA in atranslation system of interest is aminoacylated by any endogenous RS ofa translation system of interest with reduced or even zero efficiency,when compared to aminoacylation of an endogenous tRNA by an endogenousRS. In another example, an orthogonal RS aminoacylates any endogenoustRNA in the translation system of interest with reduced or even zeroefficiency, as compared to aminoacylation of the endogenous tRNA by anendogenous RS. A second orthogonal molecule can be introduced into thecell that functions with the first orthogonal molecule. For example, anorthogonal tRNA/RS pair includes introduced complementary componentsthat function together in the cell with an efficiency (e.g., about 50%efficiency, about 60% efficiency, about 70% efficiency, about 75%efficiency, about 80% efficiency, about 85% efficiency, about 90%efficiency, about 95% efficiency, or about 99% or more efficiency) tothat of a corresponding tRNA/RS endogenous pair. “Improvement inorthogonality” refers to enhanced orthogonality compared to a startingmaterial or a naturally occurring tRNA or RS.

Cognate: The term “cognate” refers to components that function together,e.g., a tRNA and an aminoacyl-tRNA synthetase. The components can alsobe referred to as being complementary.

Preferentially aminoacylates: The term “preferentially aminoacylates”refers to an efficiency, e.g., about 70% efficient, about 75% efficient,about 80% efficient, about 85% efficient, about 90% efficient, about 95%efficient, or about 99% or more efficient, at which an O-RSaminoacylates an O-tRNA with a selected amino acid, e.g., an unnaturalamino acid, compared to the O-RS aminoacylating a naturally occurringtRNA or a starting material used to generate the O-tRNA. The unnaturalamino acid is then incorporated into a growing polypeptide chain withhigh fidelity, e.g., at greater than about 70% efficiency for a givenselector codon, at greater than about 75% efficiency for a givenselector codon, at greater than about 80% efficiency for a givenselector codon, at greater than about 85% efficiency for a givenselector codon, at greater than about 90% efficiency for a givenselector codon, greater than about 95% efficiency for a given selectorcodon, or greater than about 99% efficiency for a given selector codon.

Selector codon: The term “selector codon” refers to codons recognized bythe O-tRNA in the translation process and not recognized by anendogenous tRNA. The O-tRNA anticodon loop recognizes the selector codonon the mRNA and incorporates its amino acid, e.g., a selected aminoacid, such as an unnatural amino acid, at this site in the polypeptide.Selector codons can include but are not limited to, e.g., nonsensecodons, such as, stop codons, including but not limited to, amber,ochre, and opal codons; four or more base codons; rare codons; codonsderived from natural or unnatural base pairs and/or the like. For agiven system, a selector codon can also include one of the natural threebase codons, wherein the endogenous system does not use (or rarely uses)said natural three base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem wherein the natural three base codon is a rare codon.

Suppressor tRNA: A suppressor tRNA is a tRNA that alters the reading ofa messenger RNA (mRNA) in a given translation system, e.g., by providinga mechanism for incorporating an amino acid into a polypeptide chain inresponse to a selector codon. For example, a suppressor tRNA can readthrough a codon including but not limited to, a stop codon, a four basecodon, or a rare codon.

Suppression activity: The term “suppression activity” refers to theability of a tRNA, e.g., a suppressor tRNA, to read through a selectorcodon. Activity can be expressed as a percentage of activity observed ascompared to a control (e.g., lacking a cognate synthetase).

Translation system: The term “translation system” refers to thecomponents necessary to incorporate a naturally occurring amino acidinto a growing polypeptide chain (protein). Components of a translationsystem can include, e.g., ribosomes, tRNA's, synthetases, mRNA and thelike. The components of the present invention can be added to an invitro or in vivo translation system. Examples of translation systemsinclude but are not limited to, a non-eukaryotic cell, e.g., a bacterium(such as E. coli), a eukaryotic cell, e.g., a yeast cell, a mammaliancell, a plant cell, an algae cell, a fungus cell, an insect cell, acell-free translational system e.g., a cell lysate, and/or the like.

Translation systems may be cellular or cell-free, and may be prokaryoticor eukaryotic. Cellular translation systems include, but are not limitedto, whole cell preparations such as permeabilized cells or cell cultureswherein a desired nucleic acid sequence can be transcribed to mRNA andthe mRNA translated. Cell-free translation systems are commerciallyavailable and many different types and systems are well-known. Examplesof cell-free systems include, but are not limited to, prokaryoticlysates such as Escherichia coli lysates, and eukaryotic lysates such aswheat germ extracts, insect cell lysates, rabbit reticulocyte lysates,rabbit oocyte lysates and human cell lysates. Eukaryotic extracts orlysates may be preferred when the resulting protein is glycosylated,phosphorylated or otherwise modified because many such modifications areonly possible in eukaryotic systems. Some of these extracts and lysatesare available commercially (Promega; Madison, Wis.; Stratagene; LaJolla, Calif.; Amersham; Arlington Heights, Ill.; GIBCO/BRL; GrandIsland, N.Y.). Membranous extracts, such as the canine pancreaticextracts containing microsomal membranes, are also available which areuseful for translating secretory proteins.

Reconstituted translation systems may also be used. Mixtures of purifiedtranslation factors have also been used successfully to translate mRNAinto protein as well as combinations of lysates or lysates supplementedwith purified translation factors such as initiation factor-1 (IF-1),IF-2, IF-3 (α or β), elongation factor T (EF-Tu), or terminationfactors. Cell-free systems may also be coupled transcription/translationsystems wherein DNA is introduced to the system, transcribed into mRNAand the mRNA translated as described in Current Protocols in MolecularBiology (F. M. Ausubel et al. editors, Wiley Interscience, 1993), whichis hereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(hnRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

Selected amino acid: The term “selected amino acid” refers to anydesired naturally occurring amino acid or unnatural amino acid. As usedherein, the term “unnatural amino acid” or “non-naturally encoded aminoacid” refers to any amino acid, modified amino acid, and/or amino acidanalogue that is not one of the 20 common naturally occurring aminoacids or selenocysteine or pyrrolysine. Other terms that may be usedsynonymously with the term “non-naturally encoded amino acid” and“unnatural amino acid” are “non-natural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrrolysine and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally-occurring aminoacids include, but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

Derived from: As used herein, the term “derived from” refers to acomponent that is isolated from or made using information from aspecified molecule or organism.

Positive selection or screening marker: As used herein, the term“positive selection or screening marker” refers to a marker that whenpresent, e.g., expressed, activated or the like, results inidentification of a cell with the positive selection marker from thosewithout the positive selection marker.

Negative selection or screening marker: As used herein, the term“negative selection or screening marker” refers to a marker that whenpresent, e.g., expressed, activated or the like, allows identificationof a cell that does not possess the desired property (e.g., as comparedto a cell that does possess the desired property).

Reporter: As used herein, the term “reporter” refers to a component thatcan be used to select target components of a system of interest. Forexample, a reporter can include a protein, e.g., an enzyme, that confersantibiotic resistance or sensitivity (including, but not limited to,β-lactamase, chloramphenicol acetyltransferase (CAT), and the like), afluorescent screening marker (including but not limited to, greenfluorescent protein (e.g. GFP), YFP, EGFP, RFP, a luminescent marker(including but not limited to, a firefly luciferase protein), anaffinity based screening marker, or positive or negative selectablemarker genes such as lacZ, β-gal/lacZ (β-galactosidase), ADH (alcoholdehydrogenase), his3, ura3, leu2, lys2, or the like.

Eukaryote: As used herein, the term “eukaryote” refers to organismsbelonging to the phylogenetic domain Eucarya such as animals (includingbut not limited to, mammals, insects, reptiles, birds, etc.), ciliates,plants (including but not limited to, monocots, dicots, algae, etc.),fungi, yeasts, flagellates, microsporidia, protists, etc.

Non-eukaryote: As used herein, the term “non-eukaryote” refers tonon-eukaryotic organisms. For example, a non-eukaryotic organism canbelong to the Eubacteria (including but not limited to, Escherichiacoli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonasfluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.)phylogenetic domain, or the Archaea (e.g., Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.)phylogenetic domain.

Conservative variant: The term “conservative variant” refers to atranslation component, e.g., a conservative variant O-tRNA or aconservative variant O-RS, that functionally performs like the componentfrom which the conservative variant is based, e.g., an O-tRNA or O-RS,but has variations in the sequence. For example, an O-RS willaminoacylate a complementary O-tRNA or a conservative variant O-tRNAwith a selected amino acid, e.g., an unnatural amino acid, although theO-tRNA and the conservative variant O-tRNA do not have the samesequence. Similarly, a tRNA will be aminoacylated with a selected aminoacid, e.g., an unnatural amino acid, by a complementary O-RS or aconservative variant O-RS, although the O-RS and the conservativevariant O-RS do not have the same sequence. The conservative variant canhave, e.g., one variation, two variations, three variations, fourvariations, or five or more variations in sequence, as long as theconservative variant is complementary to the corresponding O-tRNA orO-RS.

Selection or screening agent: As used herein, the term “selection orscreening agent” refers to an agent that, when present, allows for aselection/screening of certain components from a population. Forexample, a selection or screening agent includes, but is not limited to,e.g., a nutrient, an antibiotic, a wavelength of light, an antibody, anexpressed polynucleotide, or the like. The selection agent can bevaried, e.g., by concentration, intensity, etc.

The term “not efficiently recognized” refers to an efficiency, e.g.,less than about 10%, less than about 5%, or less than about 1%, at whicha RS from one organism aminoacylates O-tRNA.

DETAILED DESCRIPTION

Translation systems that are suitable for making proteins that includeone or more selected amino acids, e.g., an unnatural amino acid, aredescribed in U.S. patent application Ser. Nos. 10/126,931, entitled“METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYLtRNA SYNTHETASE PAIRS” and 10/126,927, entitled “IN VIVO INCORPORATIONOF UNNATURAL AMINO ACIDS.” In addition, see U.S. Ser. No. 10/825,867entitled “EXPANDING THE EUKARYOTIC GENETIC CODE.” Each of theseapplications is incorporated herein by reference in its entirety. Suchtranslation systems generally comprise cells that include an orthogonaltRNA (O-tRNA), an orthogonal aminoacyl tRNA synthetase (O-RS), and aselected amino acid, e.g., an unnatural amino acid, where the O-RSaminoacylates the O-tRNA with the selected amino acid. An orthogonalpair of the present invention is composed of an O-tRNA, e.g., asuppressor tRNA, a frameshift tRNA, or the like, and an O-RS. The O-tRNArecognizes a first selector codon and has suppression activity inpresence of a cognate synthetase in response to a selector codon. Thecell uses the components to incorporate the selected amino acid into agrowing polypeptide chain. For example, a nucleic acid that comprises apolynucleotide that encodes a polypeptide of interest can also bepresent, where the polynucleotide comprises a selector codon that isrecognized by the O-tRNA. The translation system can also be an in vitrosystem. RS molecules of the present invention are useful in anytranslational system, including systems that utilize ribosomes intranslation.

The translation system may also be a cell-free (in-vitro) translationalsystem. In these systems, which can include either mRNA as a template(in-vitro translation) or DNA as a template (combined in-vitrotranscription and translation), the in vitro synthesis is directed bythe ribosomes. Considerable effort has been applied to the developmentof cell-free protein expression systems. See, e.g., Kim, D. M. and J. R.Swartz, Biotechnology and Bioengineering, 74 :309-316 (2001); Kim, D. M.and J. R. Swartz, Biotechnology Letters, 22, 1537-1542, (2000); Kim, D.M., and J. R. Swartz, Biotechnology Progress, 16, 385-390, (2000); Kim,D. M., and J. R. Swartz, Biotechnology and Bioengineering, 66, 180-188,(1999); and Patnaik, R. and J. R. Swartz, Biotechniques 24, 862-868,(1998); U.S. Pat. No. 6,337,191; U.S. Patent Publication No.2002/0081660; WO 00/55353; WO 90/05785, which are incorporated byreference herein. Another approach that may be applied includes themRNA-peptide fusion technique. See, e.g., R. Roberts and J. Szostak,Proc. Natl. Acad. Sci. (USA) 94:12297-12302 (1997); A. Frankel, et al.,Chemistry & Biology 10:1043-1050 (2003). In this approach, an mRNAtemplate linked to puromycin is translated into peptide on the ribosome.If one or more tRNA molecules have been modified, non-natural aminoacids can be incorporated into the peptide as well. After the last mRNAcodon has been read, puromycin captures the C-terminus of the peptide.If the resulting mRNA-peptide conjugate is found to have interestingproperties in an in vitro assay, its identity can be easily revealedfrom the mRNA sequence. In this way, one may screen libraries ofpolypeptides comprising one or more non-naturally encoded amino acids toidentify polypeptides having desired properties. More recently, in vitroribosome translations with purified components have been reported thatpermit the synthesis of peptides substituted with non-naturally encodedamino acids. See, e.g., A. Forster et al., Proc. Natl. Acad. Sci. (USA)100:6353 (2003).

In certain embodiments, an E. coli cell comprising the RS of the presentinvention includes such a translation system. For example, the E. colicell of the present invention includes an orthogonal tRNA (O-tRNA),where the O-tRNA comprises suppression activity in presence of a cognatesynthetase in response to a selector codon; an orthogonal aminoacyl-tRNAsynthetase (O-RS); a selected amino acid; and, a nucleic acid thatcomprises a polynucleotide that encodes a polypeptide of interest, wherethe polynucleotide comprises a selector codon that is recognized by theO-tRNA.

The invention also features multiple O-tRNA/O-RS pairs in a cell, whichallows incorporation of more than one selected amino acid. In certainembodiments, the cell can further include an additional differentO-tRNA/O-RS pair and a second selected amino acid, where the O-tRNArecognizes a second selector codon and the O-RS preferentiallyaminoacylates the O-tRNA with the second selected amino acid. Forexample, a cell can further comprise, e.g., an amber suppressortRNA-aminoacyl tRNA synthetase pair derived from the tyrosyl-tRNAsynthetase of Methanococcus jannaschii.

The O-tRNA and/or the O-RS can be naturally occurring or can be derivedby mutation of a naturally occurring tRNA and/or RS, e.g., whichgenerates libraries of tRNA's and/or libraries of RSs, from a variety oforganisms. For example, one strategy of producing an orthogonaltRNA/aminoacyl-tRNA synthetase pair involves importing a heterologoustRNA/synthetase pair from, e.g., a source other than the host cell, ormultiple sources, into the host cell. The properties of the heterologoussynthetase candidate include, e.g., that it does not charge any hostcell tRNA, and the properties of the heterologous tRNA candidateinclude, e.g., that it is not aminoacylated by any host cell synthetase.In addition, the heterologous tRNA is orthogonal to all host cellsynthetases.

A second strategy for generating an orthogonal pair involves generatingmutant libraries from which to screen and/or select an O-tRNA or O-RS.These strategies can also be combined.

In various embodiments, the O-tRNA and O-RS are derived from at leastone organism. In another embodiment, the O-tRNA is derived from anaturally occurring or mutated naturally occurring tRNA from a firstorganism and the O-RS is derived from naturally occurring or mutatednaturally occurring RS from a second organism. In one embodiment, thefirst and second organisms are different. For example, an orthogonalpair may include a tRNA synthetase derived from Methanobacteriumthermoautotrophicum, and a tRNA derived from an archael tRNA (e.g., fromHalobacterium sp. NRC-1). Alternatively, the first and second organismsare the same. See the section entitled “Sources and Host Organisms”herein for additional information.

In certain embodiments of the present invention, an O-RS of the presentinvention comprises or is encoded by a polynucleotide sequence as setforth in SEQ ID NO.: 4, or a complementary polynucleotide sequencethereof, or a conservative variation thereof. In certain embodiments, anO-RS comprises an amino acid sequence as set forth in SEQ ID NO: 5. Seealso the section entitled “Nucleic Acid and Polypeptide Sequence andVariants” herein.

Orthogonal tRNA (O-tRNA)

An orthogonal tRNA (O-tRNA) mediates incorporation of a selected aminoacid into a protein that is encoded by a polynucleotide that comprises aselector codon that is recognized by the O-tRNA, e.g., in vivo or invitro. An O-tRNA may be aminoacylated with a desired amino acid by anymethod or technique, including but not limited to, chemical or enzymaticaminoacylation. The aminoacylated O-tRNA may be added directly to atranslation system. An O-tRNA may be aminoacylated by an RS of thepresent invention with a selected amino acid in vitro or in vivo. Inaddition, the RS may be an O-RS. An O-tRNA can be provided to thetranslation system (e.g., in vitro translation components, or a cell)directly, or by providing a polynucleotide that encodes an O-tRNA or aportion thereof. For example, an O-tRNA, or a portion thereof, isencoded by a polynucleotide sequence as set forth in SEQ ID NO.: 1, 2,3, or a complementary polynucleotide sequence thereof, or a conservativevariation thereof. An O-RS can be provided to the translation system(e.g., in vitro translation components, or a cell) directly (e.g. SEQ IDNO: 5 or 17) or a conservative variation thereof, or by providing apolynucleotide that encodes an O-RS or a portion thereof. For example,an O-RS, or a portion thereof, is encoded by a polynucleotide sequenceas set forth in SEQ ID NO.: 4, a polynucleotide sequence that encodesamino acid sequence SEQ ID NO: 17, or a complementary polynucleotidesequence thereof, or a conservative variation thereof.

An O-tRNA of the present invention comprises suppression activity in thepresence of a cognate synthetase in response to a selector codon.Suppression activity can be determined by any of a number of assaysknown in the art. For example, a β-galactosidase reporter assay can beused. A derivative of a plasmid that expresses lacZ gene under thecontrol of promoter is used, e.g., where the Leu-25 of the peptideVVLQRRDWEN of lacZ is replaced by a selector codon, e.g., TAG, TGA,AGGA, etc. codons, or sense codons (as a control) for tyrosine, serine,leucine, etc. The derivatived lacZ plasmid is introduced into cells froman appropriate organism (e.g., an organism where the orthogonalcomponents can be used) along with plasmid comprising an O-tRNA of thepresent invention. A cognate synthetase can also be introduced (eitheras a polypeptide or a polynucleotide that encodes the cognate synthetasewhen expressed). The cells are grown in media to a desired density,e.g., to an OD₆₀₀ of about 0.5, and β-galactosidase assays areperformed, e.g., using the BetaFluor™ β-Galactosidase Assay Kit(Novagen). Percent suppression is calculated as the percentage ofactivity for a sample relative to a comparable control, e.g., the valueobserved from the derivatived lacZ construct, where the construct has acorresponding sense codon at desired position rather than a selectorcodon.

Examples of O-tRNA's suitable for use in the present invention are anyone of the O-tRNA molecules disclosed in U.S. patent application Ser.Nos. 10/126,931, 10/126,927, and 10/825,867. In the tRNA molecule,Thymine (T) is replaced with Uracil (U). In addition, additionalmodifications to the bases can be present. The invention also includesconservative variations of O-tRNA. For example, conservative variationsof O-tRNA include those molecules that function like the O-tRNA andmaintain the tRNA L-shaped structure, but do not have the same sequence(and are other than wild type tRNA molecules). See also the sectionherein entitled “Nucleic Acid and Polypeptide Sequence and Variants.”

The composition comprising an O-tRNA can further include an orthogonalaminoacyl-tRNA synthetase (O-RS), where the O-RS preferentiallyaminoacylates the O-tRNA with a selected amino acid (e.g., an unnaturalamino acid). In certain embodiments, a composition including an O-tRNAcan further include a translation system (e.g., an in vitro or an invivo translation system). A nucleic acid comprising a polynucleotideencoding a polypeptide of interest, wherein the polynucleotide comprisesone or more selector codons recognized by the O-tRNA, or a combinationof one or more of these, can also be present in the cell or othertranslation system. See also, the section herein entitled “OrthogonalAminoacyl-tRNA Synthetases (O-RS).”

Methods of producing an orthogonal tRNA (O-tRNA), e.g., an O-tRNA, arealso a feature of the present invention. A tRNA, e.g., an O-tRNA,produced by the method is also a feature of the present invention.

Methods of producing an orthogonal tRNA include mutating the anticodonloop of each of a pool of tRNA's to allow recognition of a selectorcodon (e.g., an amber codon, an opal codon, a four base codon, etc.),thereby providing a plurality of potential O-tRNA's; and analyzingsecondary structure of a member of the plurality potential O-tRNA toidentify non-canonical base pairs in the secondary structure, andoptionally mutating the non-canonical base pairs (e.g., thenon-canonical base pairs are mutated to canonical base pairs). Thenon-canonical base pairs can be located in the stem region of thesecondary structure. An O-tRNA may possess an improvement of one or morecharacteristics or activities, such as improvement in orthogonality fora desired organism compared to the starting material, e.g., theplurality of tRNA sequences, while preserving its affinity towards adesired RS.

Alternatively, O-tRNA's may be developed by mutating a known tRNA tomodulate its interaction with or binding affinity to one or moremolecules that influence translation or are components of translationmachinery. Such components include, but are not limited to, elongationfactors. Bacterial elongation factor EF-Tu plays a key role in theelongation step in protein synthesis. Following aminoacylation of thetRNA by tRNA synthetase, EF-Tu binds the aminoacylated tRNA and bringsit to the A site of the ribosome. The ester bond between the chargedamino acid and the tRNA is protected from spontaneous hydrolysis due tothe binding between EF-Tu and aminoacylated tRNA. Stortchevoi et al.investigated mutants of the E. coli initiation tRNA^(fMet) U50:G64wobble base pair in the TΨC stem, since this base pair was found to be asecondary negative determinant blocking the tRNA's activity inelongation, presumably due to a weakened interaction between theEF-Tu.GTP and aminoacylated tRNA (JBC 2003 278(20):17672-17679). Also,LaRiviere et al. described in Science 2001 Oct. 5; 294(5540):165-8 thethermodynamic contributions of the amino acid and the tRNA body to theoverall binding affinity to EF-Tu. They indicated that the contributionsof the tRNA body and the amino acid are independent of each other andthat they compensate for one another when the tRNAs are correctlyacylated. Alterations to the interaction between EF-Tu.GTP and the tRNAaminoacylated with the unnatural amino acid may affect the efficiency ofthe loading of the tRNA to the A site of the ribosome. Potentialmutation sites may also be found by analyzing crystal structures ofcomplexes between tRNA and other components of translational machinerysuch as EF-Tu. For example, Nissen et al. have indicated that EF-Tu.GTPbinds directly to the phosphate backbone of the TΨC stem of yeastphenylalanyl-transfer RNA (Phe-tRNA) (Science 1995 270(5241):1464-1472).

The methods optionally include analyzing the homology of sequences oftRNA's and/or aminoacyl-tRNA synthetases to determine potentialcandidates for an O-tRNA, O-RS and/or pairs thereof, that appear to beorthogonal for a specific organism. Computer programs known in the artand described herein can be used for the analysis. In one example, tochoose potential orthogonal translational components for use in aprokaryotic organism, a synthetase and/or a tRNA is chosen that does notdisplay unusual homology to prokaryotic organisms.

A pool of tRNA's can also be produced by a consensus strategy. Forexample, the pool of tRNA's is produced by aligning a plurality of tRNAsequences; determining a consensus sequence; and generating a library oftRNA's using at least a portion, most of, or the entire consensussequence. For example, a consensus sequence can be compiled with acomputer program, e.g., the GCG program pileup. Optionally, degeneratepositions determined by the program are changed to the most frequentbase at those positions. A library is synthesized by techniques known inthe art using the consensus sequence. For example, overlap extension ofoligonucleotides in which each site of the tRNA gene can be synthesizedas a doped mixture of 90% the consensus sequence and 10% a mixture ofthe other 3 bases can be used to provide the library based on theconsensus sequence. Other mixtures can also be used, e.g., 75% theconsensus sequence and 25% a mixture of the other 3 bases, 80% theconsensus sequence and 20% a mixture of the other 3 bases, 95% theconsensus sequence and 5% a mixture of the other 3 bases, etc.

Libraries of mutant tRNA's can be generated using various mutagenesistechniques known in the art. For example, the mutant tRNA's can begenerated by site-specific mutations, random point mutations, homologousrecombination, DNA shuffling or other recursive mutagenesis methods,chimeric construction or any combination thereof.

Additional mutations can be introduced at a specific position(s), e.g.,at a nonconservative position(s), or at a conservative position(s), at arandomized position(s), or a combination thereof in a desired loop orregion of a tRNA, e.g., an anticodon loop, the acceptor stem, D arm orloop, variable loop, TΨC arm or loop, other regions of the tRNAmolecule, or a combination thereof. Mutations may include matched basepairs in the stem region.

Typically, an O-tRNA is obtained by subjecting to negative selection apopulation of cells of a first species, where the cells comprise amember of the plurality of potential O-tRNA's. The negative selectioneliminates cells that comprise a member of the plurality of potentialO-tRNA's that is aminoacylated by an aminoacyl-tRNA synthetase (RS) thatis endogenous to the cells. This provides a pool of tRNA's that areorthogonal to the cell of the first species.

In certain embodiments of the negative selection, a selector codon(s) isintroduced into polynucleotide that encodes a negative selection marker,e.g., an enzyme that confers antibiotic resistance, e.g., β-lactamase,an enzyme that confers a detectable product, e.g., β-galactosidase,chloramphenicol acetyltransferase (CAT), e.g., a toxic product, such asbarnase, at a non-essential position, etc. Screening/selection can bedone by growing the population of cells in the presence of a selectionagent (e.g., an antibiotic, such as ampicillin). In one embodiment, theconcentration of the selection agent is varied.

For example, to measure the activity of suppressor tRNA's, a selectionsystem is used that is based on the in vivo suppression of selectorcodon, e.g., nonsense or frameshift mutations introduced into apolynucleotide that encodes a negative selection marker, e.g., a genefor β-lactamase (bla). For example, polynucleotide variants, e.g., blavariants, with, e.g., TAG, AGGA, and TGA, at position a certainposition, are constructed. Cells, e.g., bacteria, are transformed withthese polynucleotides. In the case of an orthogonal tRNA, which cannotbe efficiently charged by endogenous E. coli synthetases, antibioticresistance, e.g., ampicillin resistance, should be about or less thanthat for a bacteria transformed with no plasmid. If the tRNA is notorthogonal, or if a heterologous synthetase capable of charging the tRNAis co-expressed in the system, a higher level of antibiotic, e.g.,ampicillin, resistance is be observed. Cells, e.g., bacteria, are chosenthat are unable to grow on LB agar plates with antibiotic concentrationsabout equal to cells transformed with no plasmids.

In the case of a toxic product (e.g., ribonuclease barnase), when amember of the plurality of potential tRNA's is aminoacylated byendogenous host, e.g., Escherichia coli synthetases (i.e., it is notorthogonal to the host, e.g., Escherichia coli synthetases), theselector codon is suppressed and the toxic polynucleotide productproduced leads to cell death. Cells harboring orthogonal tRNA ornon-functional tRNA's survive.

In one embodiment, the pool of tRNA's that are orthogonal to a desiredorganism are then subjected to a positive selection in which a selectorcodon is placed in a positive selection marker, e.g., encoded by a drugresistance gene, such a β-lactamase gene. The positive selection isperformed on cell comprising a polynucleotide encoding or comprising amember of the pool of tRNA's, a polynucleotide encoding a positiveselection marker, and a polynucleotide encoding cognate RS. Thesepolynucleotides are expressed in the cell and the cell is grown in thepresence of a selection agent, e.g., ampicillin. tRNA's are thenselected for their ability to be aminoacylated by the coexpressedcognate synthetase and to insert an amino acid in response to thisselector codon. Typically, these cells show an enhancement insuppression efficiency compared to cells harboring non-functionaltRNA's, or tRNA's that cannot efficiently be recognized by thesynthetase of interest. The cell harboring the non-functional or tRNA'sthat are not efficiently recognized by the synthetase of interest aresensitive to the antibiotic. Therefore, tRNA's that: (i) are notsubstrates for endogenous host, e.g., Escherichia coli, synthetases;(ii) can be aminoacylated by the synthetase of interest; and (iii) arefunctional in translation survive both selections.

The stringency of the selection, e.g., the positive selection, thenegative selection or both the positive and negative selection, in theabove described-methods, optionally may be varied. For example, becausebarnase is an extremely toxic protein, the stringency of the negativeselection can be controlled by introducing different numbers of selectorcodons into the barnase gene and/or by using an inducible promoter. Inanother example, the concentration of the selection or screening agentis varied (e.g., ampicillin). In one aspect, the stringency is variedbecause the desired activity can be low during early rounds. Thus, lessstringent selection criteria are applied in early rounds and morestringent criteria are applied in later rounds of selection. In certainembodiments, the negative selection, the positive selection, or both thenegative and positive selection can be repeated multiple times. Multipledifferent negative selection markers, positive selection markers or bothnegative and positive selection markers can be used. In certainembodiments, the positive and negative selection marker can be the same.

Other types of selections/screening can be used in the invention forproducing orthogonal translational components, e.g., an O-tRNA, an O-RS,and an O-tRNA/O-RS pair. For example, the negative selection marker, thepositive selection marker or both the positive and negative selectionmarkers can include a marker that fluoresces or catalyzes a luminescentreaction in the presence of a suitable reactant. In another embodiment,a product of the marker is detected by fluorescence-activated cellsorting (FACS) or by luminescence. Optionally, the marker includes anaffinity based screening marker. See, Francisco, J. A., et al., (1993)Production and fluorescence-activated cell sorting of Escherichia coliexpressing a functional antibody fragment on the external surface. ProcNatl Acad Sci USA. 90:10444-8.

Additional methods for producing a recombinant orthogonal tRNA can befound, e.g., in U.S. patent application Ser. Nos. 10/126,931, entitled“Methods and Compositions for the Production of OrthogonaltRNA-Aminoacyl tRNA Synthetase Pairs” and 10/126,127, entitled “In vivoIncorporation of Unnatural Amino Acids,” and U.S. Ser. No. 10/825,867entitled “EXPANDING THE EUKARYOTIC GENETIC CODE.” See also, Forster etal., (2003) Programming peptidomimetic synthetases by translatinggenetic codes designed de novo PNAS 100(11):6353-6357; and, Feng et al.,(2003), Expanding tRNA recognition of a tRNA synthetase by a singleamino acid change, PNAS 100(10): 5676-5681.

A tRNA may be aminoacylated with a desired amino acid by any method ortechnique, including but not limited to, chemical or enzymaticaminoacylation.

Aminoacylation may be accomplished by aminoacyl tRNA synthetases or byother enzymatic molecules, including but not limited to, ribozymes. Theterm “ribozyme” is interchangeable with “catalytic RNA.” Cech andcoworkers (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987,Concepts Biochem. 64:221-226) demonstrated the presence of naturallyoccurring RNAs that can act as catalysts (ribozymes). However, althoughthese natural RNA catalysts have only been shown to act on ribonucleicacid substrates for cleavage and splicing, the recent development ofartificial evolution of ribozymes has expanded the repertoire ofcatalysis to various chemical reactions. Studies have identified RNAmolecules that can catalyze aminoacyl-RNA bonds on their own(2′)3′-termini (Illangakekare et al., 1995 Science 267:643-647), and anRNA molecule which can transfer an amino acid from one RNA molecule toanother (Lohse et al., 1996, Nature 381:442-444).

U.S. Patent Application Publication 2003/0228593, which is incorporatedby reference herein, describes methods to construct ribozymes and theiruse in aminoacylation of tRNAs with naturally encoded and non-naturallyencoded amino acids. Substrate-immobilized forms of enzymatic moleculesthat can aminoacylate tRNAs, including but not limited to, ribozymes,may enable efficient affinity purification of the aminoacylatedproducts. Examples of suitable substrates include agarose, sepharose,and magnetic beads. The production and use of a substrate-immobilizedform of ribozyme for aminoacylation is described in Chemistry andBiology 2003, 10:1077-1084 and U.S. Patent Application Publication2003/0228593, which is incorporated by reference herein.

Chemical aminoacylation methods include, but are not limited to, thoseintroduced by Hecht and coworkers (Hecht, S. M. Acc. Chem. Res. 1992,25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C.; Chang, P.; Hecht, S. M.Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. L.; Kuroda, Y.;Kitano, S. J. Biol. Chem. 1978, 253, 4517) and by Schultz, Chamberlin,Dougherty and others (Comish, V. W.; Mendel, D.; Schultz; P. G. Angew.Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, S. A.; Ellman, J. A.;Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.;Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989,244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J.Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356,537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997,4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W.et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996,271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34), toavoid the use of synthetases in aminoacylation. Such methods or otherchemical aminoacylation methods may be used to aminoacylate tRNAmolecules.

Biosynthetic methods that employ chemically modified aminoacyl-tRNAshave been used to incorporate several biophysical probes into proteinssynthesized in vitro. See the following publications and referencescited within: Brunner, J. New Photolabeling and crosslinking methods,Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U. C., Walter, P.,Hohnson, A. E. Photocrosslinking of the signal sequence of nascentpreprolactin of the 54-kilodalton polypeptide of the signal recognitionparticle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).

Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid.See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G.A general method for site-specific incorporation of unnatural aminoacids into proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am. Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., vol. 202, 301-336(1992); and, Mendel, D., Cornish, V. W. & Schultz, P. G. Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys. BiomolStruct. 24, 435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an unnatural amino acid.Conventional site-directed mutagenesis was used to introduce the stopcodon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′-3′ Exonucleases inphosphorothioate-based olignoucleotide-directed mutagensis, NucleicAcids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the unnatural amino acid was incorporated in response to the UAGcodon which gave a protein containing that amino acid at the specifiedposition. Experiments using [³H]-Phe and experiments with α-hydroxyacids demonstrated that only the desired amino acid is incorporated atthe position specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432;and, Ellman, J. A., Mendel, D., Schultz, P. G. Site-specificincorporation of novel backbone structures into proteins, Science,255(5041):197-200 (1992).

Methods for generating catalytic RNA may involve generating separatepools of randomized ribozyme sequences, performing directed evolution onthe pools, screening the pools for desirable aminoacylation activity,and selecting sequences of those ribozymes exhibiting desiredaminoacylation activity.

Ribozymes can comprise motifs and/or regions that facilitate acylationactivity, such as a GGU motif and a U-rich region. For example, it hasbeen reported that U-rich regions can facilitate recognition of an aminoacid substrate, and a GGU-motif can form base pairs with the 3′ terminiof a tRNA. In combination, the GGU and motif and U-rich regionfacilitate simultaneous recognition of both the amino acid and tRNAsimultaneously, and thereby facilitate aminoacylation of the 3′ terminusof the tRNA.

Ribozymes can be generated by in vitro selection using a partiallyrandomized r24mini conjugated with tRNA^(Asn) _(CCCG), followed bysystematic engineering of a consensus sequence found in the activeclones. An exemplary ribozyme obtained by this method is termed “Fx3ribozyme” and is described in U.S. Pub. App. No. 2003/0228593, thecontents of which is incorporated by reference herein, acts as aversatile catalyst for the synthesis of various aminoacyl-tRNAs chargedwith cognate non-natural amino acids.

Immobilization on a substrate may be used to enable efficient affinitypurification of the aminoacylated tRNAs. Examples of suitable substratesinclude, but are not limited to, agarose, sepharose, and magnetic beads.Ribozymes can be immobilized on resins by taking advantage of thechemical structure of RNA, such as the 3′-cis-diol on the ribose of RNAcan be oxidized with periodate to yield the corresponding dialdehyde tofacilitate immobilization of the RNA on the resin. Various types ofresins can be used including inexpensive hydrazide resins whereinreductive amination makes the interaction between the resin and theribozyme an irreversible linkage. Synthesis of aminoacyl-tRNAs can besignificantly facilitated by this on-column aminoacylation technique.Kourouklis et al. Methods 2005; 36:239-4 describe a column-basedaminoacylation system.

Isolation of the aminoacylated tRNAs can be accomplished in a variety ofways. One suitable method is to elute the aminoacylated tRNAs from acolumn with a buffer such as a sodium acetate solution with 10 mM EDTA,a buffer containing 50 mMN-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid), 12.5 mM KCl,pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH 7.0).

The aminoacylated tRNAs can be added to translation reactions in orderto incorporate the amino acid with which the tRNA was aminoacylated in aposition of choice in a polypeptide made by the translation reaction.Examples of translation systems in which the aminoacylated tRNAs may beused include, but are not limited to cell lysates. Cell lysates providereaction components necessary for in vitro translation of a polypeptidefrom an input mRNA. Examples of such reaction components include but arenot limited to ribosomal proteins, rRNA, amino acids, tRNAs, GTP, ATP,translation initiation and elongation factors and additional factorsassociated with translation. Additionally, translation systems may bebatch translations or compartmentalized translation. Batch translationsystems combine reaction components in a single compartment whilecompartmentalized translation systems separate the translation reactioncomponents from reaction products that can inhibit the translationefficiency. Such translation systems are available commercially.

Further, a coupled transcription/translation system may be used. Coupledtranscription/translation systems allow for both transcription of aninput DNA into a corresponding mRNA, which is in turn translated by thereaction components. An example of a commercially available coupledtranscription/translation is the Rapid Translation System (RTS, RocheInc.). The system includes a mixture containing E. coli lysate forproviding translational components such as ribosomes and translationfactors. Additionally, an RNA polymerase is included for thetranscription of the input DNA into an mRNA template for use intranslation. RTS can use compartmentalization of the reaction componentsby way of a membrane interposed between reaction compartments, includinga supply/waste compartment and a transcription/translation compartment.

Aminoacylation of tRNA may be performed by other agents, including butnot limited to, transferases, polymerases, catalytic antibodies,multi-functional proteins, and the like.

Orthogonal Aminoacyl-tRNA Synthetases (O-RS)

An O-RS of the present invention preferentially aminoacylates an O-tRNAwith a selected amino acid in vitro or in vivo. An O-RS of the presentinvention can be provided to the translation system (e.g., in vitrotranslation components, or a cell) by a polypeptide that includes anO-RS and/or by a polynucleotide that encodes an O-RS or a portionthereof. For example, an O-RS, or a portion thereof, is encoded by apolynucleotide sequence as set forth in SEQ ID NO.: 4 or a complementarypolynucleotide sequence thereof, or a conservative variation thereof. AnO-RS of the present invention may aminoacylate a number of differentO-tRNA molecules, including but not limited to, those disclosed herein.

Methods for identifying an orthogonal aminoacyl-tRNA synthetase (O-RS),e.g., an O-RS, for use with an O-tRNA, e.g., an O-tRNA, are also afeature of the present invention. For example a method includessubjecting to positive selection a population of cells of a firstspecies, where the cells each comprise: 1) a member of a plurality ofaminoacyl-tRNA synthetases (RSs), where the plurality of RSs comprisemutant RSs, RSs derived from a species other than the first species orboth mutant RSs and RSs derived from a species other than the firstspecies; 2) the orthogonal tRNA (O-tRNA) from a second species; and 3) apolynucleotide that encodes a positive selection marker and comprises atleast one selector codon. Cells are selected or screened for those thatshow an enhancement in suppression efficiency compared to cells lackingor with a reduced amount of the member of the plurality of RSs. Cellshaving an enhancement in suppression efficiency comprise an active RSthat aminoacylates the O-tRNA. A level of aminoacylation (in vitro or invivo) by the active RS of a first set of tRNA's from the first speciesis compared to the level of aminoacylation (in vitro or in vivo) by theactive RS of a second set of tRNA's from the second species. The levelof aminoacylation can be determined by a detectable substance (e.g., alabeled amino acid or unnatural amino acid). The active RS that moreefficiently aminoacylates the second set of tRNA's compared to the firstset of tRNA's is selected, thereby providing the orthogonalaminoacyl-tRNA synthetase for use with the O-tRNA. An O-RS, e.g., anO-RS, identified by the method is also a feature of the presentinvention.

Any of a number of assays can be used to determine aminoacylation. Theseassays can be performed in vitro or in vivo. For example, in vitroaminoacylation assays are described in, e.g., Hoben, P., and Soll, D.(1985) Methods Enzymol. 113:55-59 and in U.S. Patent ApplicationPublication No. 2003/0228593. Aminoacylation can also be determined byusing a reporter along with orthogonal translation components anddetecting the reporter in a cell expressing a polynucleotide comprisingat least one selector codon that encodes a protein. See also, U.S.patent application Ser. No. 10/126,927, entitled “IN VIVO INCORPORATIONOF UNNATURAL AMINO ACIDS;” and, U.S. Ser. No. 10/825,867 entitled“EXPANDING THE EUKARYOTIC GENETIC CODE.”

An identified O-RS can be further manipulated to alter the substratespecificity of the synthetase so that only a desired unnatural aminoacid, but not any of the common 20 amino acids, are charged to theO-tRNA. Methods to generate an orthogonal aminoacyl tRNA synthetaseswith a substrate specificity for an unnatural amino acid includemutating the synthetase, e.g., at the active site in the synthetase, atthe editing mechanism site in the synthetase, at different sites bycombining different domains of synthetases, or the like, and applying aselection process. A strategy is used that is based on the combinationof a positive selection followed by a negative selection. In thepositive selection, suppression of the selector codon introduced at anon-essential position(s) of a positive marker allows cells to surviveunder positive selection pressure. In the presence of both natural andunnatural amino acids, survivors thus encode active synthetases chargingthe orthogonal suppressor tRNA with either a natural or unnatural aminoacid. In the negative selection, suppression of a selector codonintroduced at a non-essential position(s) of a negative marker removessynthetases with natural amino acid specificities. Survivors of thenegative and positive selection encode synthetases that aminoacylate(charge) the orthogonal suppressor tRNA with unnatural amino acids only.These synthetases can then be subjected to further mutagenesis, e.g.,DNA shuffling or other recursive mutagenesis methods.

The library of mutant O-RSs can be generated using various mutagenesistechniques known in the art. For example, the mutant RSs can begenerated by site-specific mutations, random point mutations, homologousrecombination, DNA shuffling or other recursive mutagenesis methods,chimeric construction or any combination thereof. For example, a libraryof mutant RSs can be produced from two or more other, e.g., smaller,less diverse “sub-libraries.” Chimeric libraries of RSs are alsoincluded in the invention. It should be noted that libraries of tRNAsynthetases from various organisms (e.g., microorganisms such aseubacteria or archaebacteria) such as libraries that comprise naturaldiversity (see, e.g., U.S. Pat. No. 6,238,884 to Short et al; U.S. Pat.No. 5,756,316 to Schallenberger et al; U.S. Pat. No. 5,783,431 toPetersen et al; U.S. Pat. No. 5,824,485 to Thompson et al; U.S. Pat. No.5,958,672 to Short et al), are optionally constructed and screened fororthogonal pairs.

Once the synthetases are subject to the positive and negativeselection/screening strategy, these synthetases can then be subjected tofurther mutagenesis. For example, a nucleic acid that encodes the O-RScan be isolated; a set of polynucleotides that encode mutated O-RSs(e.g., by random mutagenesis, site-specific mutagenesis, recombinationor any combination thereof) can be generated from the nucleic acid; and,these individual steps or a combination of these steps can be repeateduntil a mutated O-RS is obtained that preferentially aminoacylates theO-tRNA with the unnatural amino acid. In one aspect of the presentinvention, the steps are performed multiple times, e.g., at least twotimes.

Additional levels of selection/screening stringency can also be used inthe methods of the present invention, for producing O-tRNA, O-RS, orpairs thereof. The selection or screening stringency can be varied onone or both steps of the method to produce an O-RS. This could include,e.g., varying the amount of selection/screening agent that is used, etc.Additional rounds of positive and/or negative selections can also beperformed. Selecting or screening can also comprise one or more positiveor negative selection or screening that includes, e.g., a change inamino acid permeability, a change in translation efficiency, a change intranslational fidelity, etc. Typically, the one or more change is basedupon a mutation in one or more gene in an organism in which anorthogonal tRNA-tRNA synthetase pair is used to produce protein.

Other types of selections can be used in the present invention for,e.g., O-RS, O-tRNA, and O-tRNA/O-RS pair. The positive selection markercan be any of a variety of molecules including, but not limited to, aproduct that provides a nutritional supplement for growth and theselection is performed on a medium that lacks the nutritionalsupplement. Examples of polynucleotides that encode positive selectionmarkers include, but are not limited to, e.g., a reporter gene based oncomplementing the amino acid auxotrophy of a cell, a his3 gene (e.g.,where the his3 gene encodes an imidazole glycerol phosphate dehydratase,detected by providing 3-aminotriazole (3-AT)), ura3 gene, leu2 gene,lys2 gene, lacZ gene, adh gene, etc. See, e.g., G. M. Kishore, & D. M.Shah, (1988), Amino acid biosynthesis inhibitors as herbicides, AnnualReview of Biochemistry 57:627-663. In one embodiment, lacZ production isdetected by ortho-nitrophenyl-β-D-galactopyranoside (ONPG) hydrolysis.See, e.g., I. G. Serebriiskii, & E. A. Golemis, (2000), Uses of lacZ tostudy gene function: evaluation of beta-galactosidase assays employed inthe yeast two-hybrid system, Analytical Biochemistry 285:1-15.Additional positive selection markers include, e.g., luciferase, greenfluorescent protein (GFP), YFP, EGFP, RFP, the product of an antibioticresistant gene (e.g., chloramphenicol acetyltransferase (CAT)), atranscriptional modulator protein (e.g., GAL4), etc. Optionally, apolynucleotide that encodes a positive selection marker comprises aselector codon.

A polynucleotide that encodes the positive selection marker can beoperably linked to a response element. An additional polynucleotide thatencodes a transcriptional modulator protein that modulates transcriptionfrom the response element, and comprises at least one selector codon,can also be present. The incorporation of the unnatural amino acid intothe transcriptional modulator protein by the O-tRNA aminoacylated withthe unnatural amino acid results in transcription of the polynucleotide(e.g., reporter gene) encoding the positive selection marker.Optionally, the selector codon is located in or substantially near aportion of the polynucleotide that encodes a DNA binding domain of thetranscriptional modulator protein.

A polynucleotide that encodes the negative selection marker can also beoperably linked to a response element from which transcription ismediated by the transcriptional modulator protein. See, e.g., A. J.DeMaggio, et al., (2000), The yeast split-hybrid system, Method Enzymol.328:128-137; H. M. Shih, et al., (1996), A positive genetic selectionfor disrupting protein-protein interactions: identification of CREBmutations that prevent association with the coactivator CBP, Proc. Natl.Acad. Sci. U.S.A. 93:13896-13901; M. Vidal, et al., (1996), Geneticcharacterization of a mammalian protein-protein interaction domain byusing a yeast reverse two-hybrid system, Proc. Natl. Acad. Sci. U.S.A.93:10321-10326; and, M. Vidal, et al., (1996), Reverse two-hybrid andone-hybrid systems to detect dissociation of protein-protein andDNA-protein interactions (Proc. Natl. Acad. Sci. U.S.A. 93:10315-10320).The incorporation of a natural amino acid into the transcriptionalmodulator protein by the O-tRNA aminoacylated with a natural amino acidresults in transcription of the negative selection marker. Optionally,the negative selection marker comprises a selector codon. The positiveselection marker and/or negative selection marker of the invention cancomprise at least two selector codons, which each or both can compriseat least two different selector codons or at least two of the sameselector codons.

The transcriptional modulator protein is a molecule that binds (directlyor indirectly) to a nucleic acid sequence (e.g., a response element) andmodulates transcription of a sequence that is operably linked to theresponse element. A transcriptional modulator protein can be atranscriptional activator protein (e.g., GAL4, nuclear hormonereceptors, AP1, CREB, LEF/tcf family members, SMADs, VP16, SP1, etc.), atranscriptional repressor protein (e.g., nuclear hormone receptors,Groucho/tle family, Engrailed family, etc), or a protein that can haveboth activities depending on the environment (e.g., LEF/tcf, homoboxproteins, etc.). A response element is typically a nucleic acid sequencethat is recognized by the transcriptional modulator protein or anadditional agent that acts in concert with the transcriptional modulatorprotein.

Another example of a transcriptional modulator protein is thetranscriptional activator protein, GAL4. See, e.g., A. Laughon, et al.,(1984), Identification of two proteins encoded by the Saccharomycescerevisiae GAL4 gene, Molecular & Cellular Biology 4:268-275; A.Laughon, & R. F. Gesteland, (1984), Primary structure of theSaccharomyces cerevisiae GAL4 gene, Molecular & Cellular Biology4:260-267; L. Keegan, et al., (1986), Separation of DNA binding from thetranscription-activating function of a eukaryotic regulatory protein,Science 231:699-704; and, M. Ptashne, (1988), How eukaryotictranscriptional activators work, Nature 335:683-689. The N-terminal 147amino acids of this 881 amino acid protein form a DNA binding domain(DBD) that binds DNA sequence specifically. See, e.g., M. Carey, et al.,(1989), An amino-terminal fragment of GAL4 binds DNA as a dimer, J. Mol.Biol. 209:423-432; and, E. Giniger, et al., (1985), Specific DNA bindingof GAL4, a positive regulatory protein of yeast, Cell 40:767-774. TheDBD is linked, by an intervening protein sequence, to a C-terminal 113amino acid activation domain (AD) that can activate transcription whenbound to DNA. See, e.g., J. Ma, & M. Ptashne, (1987), Deletion analysisof GAL4 defines two transcriptional activating segments, Cell48:847-853: and, J. Ma, & M. Ptashne, (1987), The carboxy-terminal 30amino acids of GAL4 are recognized by GAL80, Cell 50:137-142. By placingamber codons towards, e.g., the N-terminal DBD of a single polypeptidethat contains both the N-terminal DBD of GAL4 and its C-terminal AD,amber suppression by the O-tRNA/O-RS pair can be linked totranscriptional activation by GAL4. GAL4 activated reporter genes can beused to perform both positive and negative selections with the gene.

The medium used for negative selection can comprise a selecting orscreening agent that is converted to a detectable substance by thenegative selection marker. In one aspect of the invention, thedetectable substance is a toxic substance. A polynucleotide that encodesa negative selection marker can be, e.g., an ura3 gene. For example, theURA3 reporter can be placed under control of a promoter that containsGAL4 DNA binding sites. When the negative selection marker is produced,e.g., by translation of a polynucleotide encoding the GAL4 with selectorcodons, GAL4 activates transcription of URA3. The negative selection isaccomplished on a medium that comprises 5-flubroorotic acid (5-FOA),which is converted into a detectable substance (e.g., a toxic substancewhich kills the cell) by the gene product of the ura3 gene. See, e.g.,J. D. Boeke, et al., (1984), A positive selection for mutants lackingorotidine-5′-phosphate decarboxylase activity in yeast: 5-fluorooroticacid resistance, Molecular & General Genetics 197:345-346); M. Vidal, etal., (1996), Genetic characterization of a mammalian protein-proteininteraction domain by using a yeast reverse two-hybrid system, Proc.Natl. Acad. Sci. U.S.A. 93:10321-10326; and, M. Vidal, et al., (1996),Reverse two-hybrid and one-hybrid systems to detect dissociation ofprotein-protein and DNA-protein interactions, Proc. Natl. Acad. Sci.U.S.A. 93:10315-10320.

As with the positive selection marker, the negative selection marker canalso be any of a variety of molecules. The positive selection markerand/or the negative selection marker may be a polypeptide thatfluoresces or catalyzes a luminescent reaction in the presence of asuitable reactant. For example, negative selection markers include, butare not limited to, e.g., luciferase, green fluorescent protein (GFP),YFP, EGFP, RFP, the product of an antibiotic resistant gene (e.g.,chloramphenicol acetyltransferase (CAT)), the product of a lacZ gene,transcriptional modulator protein, etc. The positive selection markerand/or the negative selection marker may be detected byfluorescence-activated cell sorting (FACS) or by luminescence. Thepositive selection marker and/or negative selection marker may comprisean affinity based screening marker. The same polynucleotide can encodeboth the positive selection marker and the negative selection marker.For example, the positive selection step, the negative selection step orboth the positive and negative selection steps and can include using areporter, wherein the reporter is detected by fluorescence-activatedcell sorting (FACS). For example, a positive selection can be done firstwith a positive selection marker, e.g., chloramphenicolacetyltransferase (CAT) gene, where the CAT gene comprises a selectorcodon, e.g., an amber stop codon, in the CAT gene, which followed by anegative selection screen, that is based on the inability to suppress aselector codon(s), e.g., two or more, at positions within a negativemarker, e.g., T7 RNA polymerase gene. The positive selection marker andthe negative selection marker can be found on the same vector, e.g.,plasmid. Expression of the negative marker drives expression of thereporter, e.g., green fluorescent protein (GFP). The stringency of theselection and screen can be varied, e.g., the intensity of the lightneed to fluorescence the reporter can be varied. A positive selectioncan be done with a reporter as a positive selection marker, which isscreened by FACS, followed by a negative selection screen, that is basedon the inability to suppress a selector codon(s), e.g., two or more, atpositions within a negative marker, e.g., barnase gene.

Optionally, the reporter is displayed on a cell surface, e.g., on aphage display or the like. Cell-surface display, e.g., the OmpA-basedcell-surface display system, relies on the expression of a particularepitope, e.g., a poliovirus C3 peptide fused to an outer membrane porinOmpA, on the surface of the Escherichia coli cell. The epitope isdisplayed on the cell surface only when a selector codon in the proteinmessage is suppressed during translation. The displayed peptide thencontains the amino acid recognized by one of the mutant aminoacyl-tRNAsynthetases in the library, and the cell containing the correspondingsynthetase gene can be isolated with antibodies raised against peptidescontaining specific unnatural amino acids. The OmpA-based cell-surfacedisplay system was developed and optimized by Georgiou et al. as analternative to phage display. See, Francisco, J. A., Campbell, R.,Iverson, B. L. & Georgoiu, G. Production and fluorescence-activated cellsorting of Escherichia coli expressing a functional antibody fragment onthe external surface. Proc. Natl. Acad. Sci. USA 90:10444-8 (1993).

Other embodiments of the present invention include carrying one or moreof the selection steps in vitro. The selected component, e.g.,synthetase and/or tRNA, can then be introduced into a cell for use in invivo incorporation of an unnatural amino acid.

Additional details for producing O-RS, and altering the substratespecificity of the synthetase can be found in U.S. patent applicationSer. No. 10/126,931 entitled “Methods and Compositions for theProduction of Orthogonal tRNA-Aminoacyl tRNA Synthetase Pairs” and, U.S.Ser. No. 10/825,867 entitled “EXPANDING THE EUKARYOTIC GENETIC CODE,”which are incorporated by reference herein. Additional details forproducing O-RS can be found in Hamano-Takaku et al., (2000) A mutantEscherichia coli Tyrosyl-tRNA Synthetase Utilizes the Unnatural AminoAcid Azatyrosine More Efficiently than Tyrosine, Journal of BiologicalChemistry, 275(51):40324-40328; Kiga et al. (2002), An engineeredEscherichia coli tyrosyl-tRNA synthetase for site-specific incorporationof an unnatural amino acid into proteins in eukaryotic translation andits application in a wheat germ cell-free system, PNAS 99(15):9715-9723; and, Francklyn et al., (2002), Aminoacyl-tRNA synthetases:Versatile players in the changing theater of translation; RNA,8:1363-1372, each of which are incorporated by reference herein.

Source and Host Organisms

The translational components of the present invention are typicallyderived from non-eukaryotic organisms. For example, the orthogonalO-tRNA can be derived from a non-eukaryotic organism, e.g., anarchaebacterium, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium such as Haloferax volcanii andHalobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcusfuriosus, Pyrococcus horikoshii, Aeuropyrum pernix, or the like, or aeubacterium, such as Escherichia coli, Thermus thermophilus, Bacillusstearothermphilus, or the like, while the orthogonal O-RS can be derivedfrom a non-eukaryotic organism, e.g., Methanobacteriumthermoautotrophicum, Halobacterium such as Haloferax volcanii andHalobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcusfuriosus, Pyrococcus horikoshii, Aeuropyrum pernix, or the like, or aeubacterium, such as Escherichia coli, Thermus thermophilus, Bacillusstearothermphilus, or the like. In one embodiment, eukaryotic sourcescan also be used, including but not limited to, plants, algae, protists,fungi, yeasts, animals (e.g., mammals, insects, arthropods, etc.), orthe like.

The individual components of an O-tRNA/O-RS pair can be derived from thesame organism or different organisms. In one embodiment, the O-tRNA/O-RSpair is from the same organism. Alternatively, the O-tRNA and the O-RSof the O-tRNA/O-RS pair are from different organisms. For example, theO-tRNA can be derived from, e.g., a Halobacterium sp NRC-1, and the O-RScan be derived from, e.g., a Methanobacterium thermoautrophicum.

The O-tRNA, O-RS or O-tRNA/O-RS pair can be selected or screened in vivoor in vitro and/or used in a cell, e.g., a non-eukaryotic cells (such asE. coli cell), or a eukaryotic cell, to produce a polypeptide with aselected amino acid (e.g., an unnatural amino acid). A non-eukaryoticcell can be from a variety of sources, such as the Archaea phylogeneticdomain, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, or thelike, or can belong to the Eubacteria phylogenetic domain (including butnot limited to, Escherichia coli, Thermus thermophilus, Bacillusstearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa,Pseudomonas putida, or the like. A eukaryotic cell can be from a varietyof sources, including but not limited to, a plant (e.g., complex plantsuch as monocots, or dicots), an algae, a protist, a fungus, a yeast(including but not limited to, Saccharomyces cerevisiae), an animal(including but not limited to, a mammal, an insect, an arthropod, etc.),or the like. Compositions of cells with translational components of thepresent invention are also a feature of the present invention. See alsoU.S. Ser. No. 10/825,867 entitled “Expanding the Eukaryotic GeneticCode” for screening O-tRNA and/or O-RS in one species for use in anotherspecies.

To express a polypeptide of interest with a selected amino acid in ahost cell, one may subclone polynucleotides encoding a polypeptide ofinterest into an expression vector that contains a promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.

Bacterial expression systems for expressing a polypeptide of interestare available in, including but not limited to, E. coli, Bacillus sp.,Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, andSalmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature302:543-545 (1983)). Kits for such expression systems are commerciallyavailable. Eukaryotic expression systems for mammalian cells, yeast, andinsect cells are well known in the art and are also commerciallyavailable.

A tRNA and/or RS of the present invention and/or a polypeptide ofinterest may be utilized and/or expressed in any number of suitableexpression systems including, for example, yeast, insect cells,mammalian cells, and bacteria. A description of exemplary expressionsystems is provided below.

Yeast As used herein, the term “yeast” includes any of the variousyeasts capable of expressing a polypeptide of interest. Such yeastsinclude, but are not limited to, ascosporogenous yeasts (Endomycetales),basidiosporogenous yeasts and yeasts belonging to the Fungi imperfecti(Blastomycetes) group. The ascosporogenous yeasts are divided into twofamilies, Spermophthoraceae and Saccharomycetaceae. The latter iscomprised of four subfamilies, Schizosaccharomycoideae (e.g., genusSchizosaccharomyces), Nadsonioideae, Lipomycoideae and Saccharomycoideae(e.g., genera Pichia, Kluyveromyces and Saccharomyces). Thebasidiosporogenous yeasts include the genera Leucosporidium,Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeastsbelonging to the Fungi Imperfecti (Blastomycetes) group are divided intotwo families, Sporobolomycetaceae (e.g., genera Sporobolomyces andBullera) and Cryptococcaceae (e.g., genus Candida).

Of particular interest for use with the present invention are specieswithin the genera Pichia, Kluyveromyces, Saccharomyces,Schizosaccharomyces, Hansenula, Torulopsis, and Candida, including, butnot limited to, P. pastoris, P. guillerimondii, S. cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S, norbensis,S. oviformis, K. lactis, K fragilis, C. albicans, C. maltosa, and H.polymorpha. Yeast are generally available from a variety of sourcesincluding, but not limited to, the Yeast Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.), and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

The term “yeast host” or “yeast host cell” includes yeast that can be,or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original yeast hostcell that has received the recombinant vectors or other transfer DNA. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a polypeptide of interest,are included in the progeny intended by this definition.

Expression and transformation vectors, including extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeast hosts. For example, expression vectors have beendeveloped for S. cerevisiae (Sikorski et al., GENETICS (1989) 122:19;Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC. NATL.ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL.BIOL. (1986) 6:142); C. maltosa (Kunze et al., J. BASIC MICROBIOL.(1985) 25:141); H. polymorpha (Gleeson et al., J. GEN. MICROBIOL. (1986)132:3459; Roggenkamp et al., MOL. GENETICS AND GENOMICS (1986) 202:302);K. fragilis (Das et al., J. BACTERIOL. (1984) 158:1165); K. lactis (DeLouvencourt et al., J. BACTERIOL. (1983) 154:737; Van den Berg et al.,BIOTECHNOLOGY (NY) (1990) 8:135); P. guillerimondii (Kunze et al., J.BASIC MICROBIOL. (1985) 25:141); P. pastoris (U.S. Pat. Nos. 5,324,639;4,929,555; and 4,837,148; Cregg et al., MOL. CELL. BIOL. (1985) 5:3376);Schizosaccharomyces pombe (Beach et al., NATURE (1982) 300:706); and Y.lipolytica; A. nidulans (Ballance et al., BIOCHEM. BIOPHYS. RES. COMMUN.(1983) 112:284-89; Tilburn et al., GENE (1983) 26:205-221; and Yelton etal., PROC. NATL. ACAD. SCI. USA (1984) 81:1470-74); A. niger (Kelly andHynes, EMBO J. (1985) 4:475-479); T. reesia (EP 0 244 234); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357), each incorporated by reference herein.

Control sequences for yeast vectors are known to those of ordinary skillin the art and include, but are not limited to, promoter regions fromgenes such as alcohol dehydrogenase (ADH) (EP 0 284 044); enolase;glucokinase; glucose-6-phosphate isomerase;glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase;phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase(PyK) (EP 0 329 203). The yeast PHO5 gene, encoding acid phosphatase,also may provide useful promoter sequences (Miyanohara et al., PROC.NATL. ACAD. SCI. USA (1983) 80:1). Other suitable promoter sequences foruse with yeast hosts may include the promoters for 3-phosphoglyceratekinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255:12073); and otherglycolytic enzymes, such as pyruvate decarboxylase, triosephosphateisomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY(1978) 17:4900; Hess et al., J. ADV. ENZYME REG. (1969) 7:149).Inducible yeast promoters having the additional advantage oftranscription controlled by growth conditions may include the promoterregions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase;metallothionein; glyceraldehyde-3-phosphate dehydrogenase; degradativeenzymes associated with nitrogen metabolism; and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 0 073 657.

Yeast enhancers also may be used with yeast promoters. In addition,synthetic promoters may also function as yeast promoters. For example,the upstream activating sequences (UAS) of a yeast promoter may bejoined with the transcription activation region of another yeastpromoter, creating a synthetic hybrid promoter. Examples of such hybridpromoters include the ADH regulatory sequence linked to the GAPtranscription activation region. See U.S. Pat. Nos. 4,880,734 and4,876,197, which are incorporated by reference herein. Other examples ofhybrid promoters include promoters that consist of the regulatorysequences of the ADH2, GAL4, GAL10, or PHO5 genes, combined with thetranscriptional activation region of a glycolytic enzyme gene such asGAP or PyK. See EP 0 164 556. Furthermore, a yeast promoter may includenaturally occurring promoters of non-yeast origin that have the abilityto bind yeast RNA polymerase and initiate transcription.

Other control elements that may comprise part of the yeast expressionvectors include terminators, for example, from GAPDH or the enolasegenes (Holland et al., J. BIOL. CHEM. (1981) 256:1385). In addition, theorigin of replication from the 2μ plasmid origin is suitable for yeast.A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid. See Tschumper et al., GENE (1980) 10:157; Kingsman etal., GENE (1979) 7:141. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

Methods of introducing exogenous DNA into yeast hosts are known to thoseof ordinary skill in the art, and typically include, but are not limitedto, either the transformation of spheroplasts or of intact yeast hostcells treated with alkali cations. For example, transformation of yeastcan be carried out according to the method described in Hsiao et al.,PROC. NATL. ACAD. SCI. USA (1979) 76:3829 and Van Solingen et al., J.BACT. (1977) 130:946. However, other methods for introducing DNA intocells such as by nuclear injection, electroporation, or protoplastfusion may also be used as described generally in SAMBROOK ET AL.,MOLECULAR CLONING: A LAB. MANUAL (2001). Yeast host cells may then becultured using standard techniques known to those of ordinary skill inthe art.

Other methods for expressing heterologous proteins in yeast host cellsare known to those of ordinary skill in the art. See generally U.S.Patent Publication No. 20020055169, U.S. Pat. Nos. 6,361,969; 6,312,923;6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; and5,089,398; U.S. Reexamined Pat. Nos. RE37,343 and RE35,749; PCTPublished Patent Applications WO 99/07862; WO 98/37208; and WO 98/26080;European Patent Applications EP 0 946 736; EP 0 732 403; EP 0 480 480;WO 90/10277; EP 0 340 986; EP 0 329 203; EP 0 324 274; and EP 0 164 556.See also Gellissen et al., ANTONIE VAN LEEUWENHOEK (1992) 62(1-2):79-93;Romanos et al., YEAST (1992) 8(6):423-488; Goeddel, METHODS INENZYMOLOGY (1990) 185:3-7, each incorporated by reference herein.

The yeast host strains may be grown in fermentors during theamplification stage using standard feed batch fermentation methods knownto those of ordinary skill in the art. The fermentation methods may beadapted to account for differences in a particular yeast host's carbonutilization pathway or mode of expression control. For example,fermentation of a Saccharomyces yeast host may require a single glucosefeed, complex nitrogen source (e.g., casein hydrolysates), and multiplevitamin supplementation. In contrast, the methylotrophic yeast P.pastoris may require glycerol, methanol, and trace mineral feeds, butonly simple ammonium (nitrogen) salts for optimal growth and expression.See, e.g., U.S. Pat. No. 5,324,639; Elliott et al., J. PROTEIN CHEM.(1990) 9:95; and Fieschko et al., BIOTECH. BIOENG. (1987) 29:1113,incorporated by reference herein.

Such fermentation methods, however, may have certain common featuresindependent of the yeast host strain employed. For example, a growthlimiting nutrient, typically carbon, may be added to the fermentorduring the amplification phase to allow maximal growth. In addition,fermentation methods generally employ a fermentation medium designed tocontain adequate amounts of carbon, nitrogen, basal salts, phosphorus,and other minor nutrients (vitamins, trace minerals and salts, etc.).Examples of fermentation media suitable for use with Pichia aredescribed in U.S. Pat. Nos. 5,324,639 and 5,231,178, which areincorporated by reference herein.

Baculovirus-Infected Insect Cells The term “insect host” or “insect hostcell” refers to a insect that can be, or has been, used as a recipientfor recombinant vectors or other transfer DNA. The term includes theprogeny of the original insect host cell that has been transfected. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a polypeptide of interest,are included in the progeny intended by this definition.

The selection of suitable insect cells for expression of a polypeptideof interest is known to those of ordinary skill in the art. Severalinsect species are well described in the art and are commerciallyavailable including Aedes aegypti, Bombyx mori, Drosophila melanogaster,Spodoptera frugiperda, and Trichoplusia ni. In selecting insect hostsfor expression, suitable hosts may include those shown to have, interalia, good secretion capacity, low proteolytic activity, and overallrobustness. Insect are generally available from a variety of sourcesincluding, but not limited to, the Insect Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.); and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

Generally, the components of a baculovirus-infected insect expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene to be expressed;a wild type baculovirus with sequences homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.The materials, methods and techniques used in constructing vectors,transfecting cells, picking plaques, growing cells in culture, and thelike are known in the art and manuals are available describing thesetechniques.

After inserting the heterologous gene into the transfer vector, thevector and the wild type viral genome are transfected into an insecthost cell where the vector and viral genome recombine. The packagedrecombinant virus is expressed and recombinant plaques are identifiedand purified. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, forexample, Invitrogen Corp. (Carlsbad, Calif.). These techniques aregenerally known to those of ordinary skill in the art and fullydescribed in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATIONBULLETIN No. 1555 (1987), herein incorporated by reference. See also,RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSIONPROTOCOLS (1995); AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY16.9-16.11 (1994); KING AND POSSEE, THE BACULOVIRUS SYSTEM: A LABORATORYGUIDE (1992); and O'REILLY ET AL., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992).

Indeed, the production of various heterologous proteins usingbaculovirus/insect cell expression systems is known to those of ordinaryskill in the art. See, e.g., U.S. Pat. Nos. 6,368,825; 6,342,216;6,338,846; 6,261,805; 6,245,528, 6,225,060; 6,183,987; 6,168,932;6,126,944; 6,096,304; 6,013,433; 5,965,393; 5,939,285; 5,891,676;5,871,986; 5,861,279; 5,858,368; 5,843,733; 5,762,939; 5,753,220;5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032; WO99/51721; WO 99/45130; WO 99/31257; WO 99/10515; WO 99/09193; WO97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO93/03173; WO 92/16619; WO 92/02628; WO 92/01801; WO 90/14428; WO90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO89/01037; WO 88/07082, which are incorporated by reference herein.

Vectors that are useful in baculovirus/insect cell expression systemsare known in the art and include, for example, insect expression andtransfer vectors derived from the baculovirus Autographacalifornicanuclear polyhedrosis virus (AcNPV), which is a helper-independent, viralexpression vector. Viral expression vectors derived from this systemusually use the strong viral polyhedrin gene promoter to driveexpression of heterologous genes. See generally, O'Reilly ET AL.,BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Prior to inserting the foreign gene into the baculovirus genome, theabove-described components, comprising a promoter, leader (if desired),coding sequence of interest, and transcription termination sequence, aretypically assembled into an intermediate transplacement construct(transfer vector). Intermediate transplacement constructs are oftenmaintained in a replicon, such as an extra chromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as bacteria. Thereplicon will have a replication system, thus allowing it to bemaintained in a suitable host for cloning and amplification. Morespecifically, the plasmid may contain the polyhedrin polyadenylationsignal (Miller, ANN. REV. MICROBIOL. (1988) 42:177) and a prokaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

One commonly used transfer vector for introducing foreign genes intoAcNPV is pAc373. Many other vectors, known to those of skill in the art,have also been designed including, for example, pVL985, which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 base pairs downstream from the ATT. See Luckow andSummers, VIROLOGY 170:31 (1989). Other commercially available vectorsinclude, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac;pBlueBac4.5 (Invitrogen Corp., Carlsbad, Calif.).

After insertion of the heterologous gene, the transfer vector and wildtype baculoviral genome are co-transfected into an insect cell host.Methods for introducing heterologous DNA into the desired site in thebaculovirus virus are known in the art. See SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN No. 1555 (1987); Smith et al.,MOL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989)170:31. For example, the insertion can be into a gene such as thepolyhedrin gene, by homologous double crossover recombination; insertioncan also be into a restriction enzyme site engineered into the desiredbaculovirus gene. See Miller et al., BIOESSAYS (1989) 11(4):91.

Transfection may be accomplished by electroporation. See TROTTER ANDWOOD, 39 METHODS IN M OLECULAR BIOLOGY (1995); Mann and King, J. GEN.VIROL. (1989) 70:3501. Alternatively, liposomes may be used to transfectthe insect cells with the recombinant expression vector and thebaculovirus. See, e.g., Liebman et al., BIOTECHNIQUES (1999) 26(1):36;Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL.CHEM. (1998) 273(22):13570; Schmidt et al., PROTEIN EXPRESSION ANDPURIFICATION (1998) 12:323; Siffert et al., NATURE GENETICS (1998)18:45; TILKINS ET AL., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154(1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263;Dolphin et al., NATURE GENETICS (1997) 17:491; Kost et al., GENE (1997)190:139; Jakobsson et al., J. BIOL. CHEM. (1996) 271:22203; Rowles etal., J. BIOL. CHEM. (1996) 271(37):22376; Reverey et al., J. BIOL. CHEM.(1996) 271(39):23607-10; Stanley et al., J. BIOL. CHEM. (1995) 270:4121;Sisk et al., J. VIROL. (1994) 68(2):766; and Peng et al., BIOTECHNIQUES(1993) 14(2):274. Commercially available liposomes include, for example,Cellfectin® and Lipofectin® (Invitrogen, Corp., Carlsbad, Calif.). Inaddition, calcium phosphate transfection may be used. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990)18(19):5667; and Mann and King, J. GEN. VIROL. (1989) 70:3501.

Baculovirus expression vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g., structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus promoter may also have asecond domain called an enhancer, which, if present, is usually distalto the structural gene. Moreover, expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in the infectioncycle, provide particularly useful promoter sequences. Examples includesequences derived from the gene encoding the viral polyhedron protein(FRIESEN ET AL., The Regulation of Baculovirus Gene Expression in T HEMOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476)and the gene encoding the p10 protein (Vlak et al., J. GEN. VIROL.(1988) 69:765).

The newly formed baculovirus expression vector is packaged into aninfectious recombinant baculovirus and subsequently grown plaques may bepurified by techniques known to those of ordinary skill in the art. SeeMiller et al., BIOESSAYS (1989) 11(4):91; SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia, Aedes aegypti (ATCCNo. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster(ATCC No. 1963), Spodoptera frugiperda, and Trichoplusia ni. See Wright,NATURE (1986) 321:718; Carbonell et al., J. VIROL. (1985) 56:153; Smithet al., MOL. CELL. BIOL. (1983) 3:2156. See generally, Fraser et al., INVITRO CELL. DEV. BIOL. (1989) 25:225. More specifically, the cell linesused for baculovirus expression vector systems commonly include, but arenot limited to, SD (Spodoptera frugiperda) (ATCC No. CRL-1711), Sf21(Spodoptera frugiperda) (Invitrogen Corp., Cat. No. 11497-013 (Carlsbad,Calif.)), Tri-368 (Trichopulsia ni), and High-Five™ BTI-TN-5B1-4(Trichopulsia ni).

Cells and culture media are commercially available for both direct andfusion expression of heterologous polypeptides in abaculovirus/expression, and cell culture technology is generally knownto those of ordinary skill in the art.

E. Coli, Pseudomonas species, and other Prokaryotes Bacterial expressiontechniques are known to those of ordinary skill in the art. A widevariety of vectors are available for use in bacterial hosts. The vectorsmay be single copy or low or high multicopy vectors. Vectors may servefor cloning and/or expression. In view of the ample literatureconcerning vectors, commercial availability of many vectors, and evenmanuals describing vectors and their restriction maps andcharacteristics, no extensive discussion is required here. As iswell-known, the vectors normally involve markers allowing for selection,which markers may provide for cytotoxic agent resistance, prototrophy orimmunity. Frequently, a plurality of markers is present, which providefor different characteristics.

A bacterial promoter is any DNA sequence capable of binding bacterialRNA polymerase and initiating the downstream (3′) transcription of acoding sequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site and a transcriptioninitiation site. A bacterial promoter may also have a second domaincalled an operator that may overlap an adjacent RNA polymerase bindingsite at which RNA synthesis begins. The operator permits negativeregulated (inducible) transcription, as a gene repressor protein maybind the operator and thereby inhibit transcription of a specific gene.Constitutive expression may occur in the absence of negative regulatoryelements, such as the operator. In addition, positive regulation may beachieved by a gene activator protein binding sequence, which, if presentis usually proximal (5′) to the RNA polymerase binding sequence. Anexample of a gene activator protein is the catabolite activator protein(CAP), which helps initiate transcription of the lac operon inEscherichia coli (E. coli) [Raibaud et al., ANNU. REV. GENET. (1984)18:173]. Regulated expression may therefore be either positive ornegative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal., NATURE (1977) 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al., Nuc. ACIDS RES. (1980) 8:4057; Yelverton et al.,NUCL. ACIDS RES. (1981) 9:731; U.S. Pat. No. 4,738,921; EP Pub. Nos. 036776 and 121 775, which are incorporated by reference herein]. Theβ-galactosidase (bla) promoter system [Weissmann (1981) “The cloning ofinterferon and other mistakes.” In Interferon 3 (Ed. I. Gresser)],bacteriophage lambda PL [Shimatake et al., NATURE (1981) 292:128] and T5[U.S. Pat. No. 4,689,406, which are incorporated by reference herein]promoter systems also provide useful promoter sequences. Strongpromoters, such as the T7 promoter may be used to induce the polypeptideof interest at high levels. Examples of such vectors are known to thoseof ordinary skill in the art and include the pET29 series from Novagen,and the pPOP vectors described in WO99/05297, which is incorporated byreference herein. Such expression systems produce high levels ofpolypeptide in the host without compromising host cell viability orgrowth parameters. pET19 (Novagen) is another vector known in the art.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433, which isincorporated by reference herein]. For example, the tac promoter is ahybrid trp-lac promoter comprised of both trp promoter and lac operonsequences that is regulated by the lac repressor [Amann et al., GENE(1983) 25:167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80:21].Furthermore, a bacterial promoter can include naturally occurringpromoters of non-bacterial origin that have the ability to bindbacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al., J.MOL. BIOL. (1986) 189:113; Tabor et al., Proc Natl. Acad. Sci. (1985)82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EP Pub. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al., NATURE (1975) 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. “Genetic signals and nucleotide sequences inmessenger RNA”, In Biological Regulation and Development: GeneExpression (Ed. R. F. Goldberger, 1979)]. To express eukaryotic genesand prokaryotic genes with weak ribosome-binding site [Sambrook et al.“Expression of cloned genes in Escherichia coli”, Molecular Cloning: ALaboratory Manual, 1989].

The term “bacterial host” or “bacterial host cell” refers to a bacterialthat can be, or has been, used as a recipient for recombinant vectors orother transfer DNA. The term includes the progeny of the originalbacterial host cell that has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement to theoriginal parent, due to accidental or deliberate mutation. Progeny ofthe parental cell that are sufficiently similar to the parent to becharacterized by the relevant property, such as the presence of anucleotide sequence encoding a polypeptide of interest, are included inthe progeny intended by this definition.

The selection of suitable host bacteria for expression of polypeptidesis known to those of ordinary skill in the art. In selecting bacterialhosts for expression, suitable hosts may include those shown to have,inter alia, good inclusion body formation capacity, low proteolyticactivity, and overall robustness. Bacterial hosts are generallyavailable from a variety of sources including, but not limited to, theBacterial Genetic Stock Center, Department of Biophysics and MedicalPhysics, University of California (Berkeley, Calif.); and the AmericanType Culture Collection (“ATCC”) (Manassas, Va.).Industrial/pharmaceutical fermentation generally use bacterial derivedfrom K strains (e.g. W3110) or from bacteria derived from B strains(e.g. BL21). These strains are particularly useful because their growthparameters are extremely well known and robust. In addition, thesestrains are non-pathogenic, which is commercially important for safetyand environmental reasons. Other examples of suitable E. coli hostsinclude, but are not limited to, strains of BL21, DH10B, or derivativesthereof. In another embodiment of the methods of the present invention,the E. coli host is a protease minus strain including, but not limitedto, OMP- and LON-. The host cell strain may be a species of Pseudomonas,including but not limited to, Pseudomonas fluorescens, Pseudomonasaeruginosa, and Pseudomonas putida. Pseudomonas fluorescens biovar 1,designated strain MB101, is known to be useful for recombinantproduction and is available for therapeutic protein productionprocesses. Examples of a Pseudomonas expression system include thesystem available from The Dow Chemical Company as a host strain(Midland, Mich. available on the World Wide Web at dow.com). U.S. Pat.Nos. 4,755,465 and 4,859,600, which are incorporated by referenceherein, describe the use of Pseudomonas strains as a host cell for hGHproduction.

Once a recombinant host cell strain has been established (i.e., theexpression construct has been introduced into the host cell and hostcells with the proper expression construct are isolated), therecombinant host cell strain is cultured under conditions appropriatefor production of the polypeptide of interest. As will be apparent toone of skill in the art, the method of culture of the recombinant hostcell strain will be dependent on the nature of the expression constructutilized and the identity of the host cell. Recombinant host strains arenormally cultured using methods that are well known to the art.Recombinant host cells are typically cultured in liquid mediumcontaining assimilatable sources of carbon, nitrogen, and inorganicsalts and, optionally, containing vitamins, amino acids, growth factors,and other proteinaceous culture supplements known to those of ordinaryskill in the art. Liquid media for culture of host cells may optionallycontain antibiotics or anti-fungals to prevent the growth of undesirablemicroorganisms and/or compounds including, but not limited to,antibiotics to select for host cells containing the expression vector.

Recombinant host cells may be cultured in batch or continuous formats,with either cell harvesting (in the case where the polypeptide ofinterest accumulates intracellularly) or harvesting of culturesupernatant in either batch or continuous formats. For production inprokaryotic host cells, batch culture and cell harvest are preferred.

Selector Codons

Selector codons of the present invention expand the genetic codonframework of protein biosynthetic machinery. For example, a selectorcodon includes, e.g., a unique three base codon, a nonsense codon, suchas a stop codon, including but not limited to, an amber codon (UAG), anochre codon, or an opal codon (UGA), an unnatural codon, a four base (ormore) codon, a rare codon, or the like. A number of selector codons canbe introduced into a desired gene or polynucleotide, e.g., one or more,two or more, three or more, etc.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of a selected amino acid, e.g., anunnatural amino acid, in vivo. For example, an O-tRNA is produced thatrecognizes the stop codon and is aminoacylated by an O-RS with aselected amino acid. This O-tRNA is not recognized by the naturallyoccurring host's aminoacyl-tRNA synthetases. Conventional site-directedmutagenesis can be used to introduce the stop codon at the site ofinterest in a polypeptide of interest. See; e.g., Sayers, J. R., et al.(1988), 5′-3′ Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis. Nucleic Acids Res, 16:791-802.When the O-RS, O-tRNA and the nucleic acid that encodes a polypeptide ofinterest are combined, e.g., in vivo, the selected amino acid isincorporated in response to the stop codon to give a polypeptidecontaining the selected amino acid, e.g., an unnatural amino acid, atthe specified position. In one embodiment of the present invention, astop codon used as a selector codon is an amber codon, UAG, and/or anopal codon, UGA. For example, see SEQ ID NO.: 6 for an example of anO-tRNA that recognizes an amber codon, and see SEQ ID NO.: 7 for anexample of an O-tRNA that recognizes an opal codon. A genetic code inwhich UAG and UGA are both used as a selector codon can encode 22 aminoacids while preserving the ochre nonsense codon, UAA, which is the mostabundant termination signal.

The incorporation of selected amino acids, e.g., unnatural amino acids,in vivo can be done without significant perturbation of the host cell.For example in non-eukaryotic cells, such as Escherichia coli, becausethe suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, e.g., the amber suppressor tRNA, and therelease factor 1 (RF1) (which binds to the UAG codon and initiatesrelease of the growing peptide from the ribosome), the suppressionefficiency can be modulated by, e.g., either increasing the expressionlevel of O-tRNA, e.g., the suppressor tRNA, or using an RF1 deficientstrain. In eukaryotic cells, because the suppression efficiency for theUAG codon depends upon the competition between the O-tRNA, e.g., theamber suppressor tRNA, and a eukaryotic release factor (e.g., eRF)(which binds to a stop codon and initiates release of the growingpeptide from the ribosome), the suppression efficiency can be modulatedby, e.g., increasing the expression level of O-tRNA, e.g., thesuppressor tRNA.

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In thiscase, the synthetic tRNA competes with the naturally occurring tRNAArg,which exists as a minor species in Escherichia coli. Some organisms donot use all triplet codons. An unassigned codon AGA in Micrococcusluteus has been utilized for insertion of amino acids in an in vitrotranscription/translation extract. See, e.g., Kowal and Oliver, Nucl.Acid. Res., 25:4685 (1997). Components of the present invention can begenerated to use these rare codons in vivo.

Selector codons also comprise extended codons, e.g., four or more basecodons, such as, four, five, six or more base codons. Examples of fourbase codons include but are not limited to, AGGA, CUAG, UAGA, CCCU, andthe like. Examples of five base codons include but are not limited to,AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like. A feature mayinclude using extended codons based on frameshift suppression. Four ormore base codons can insert, e.g., one or multiple selected amino acids,including but not limited to, unnatural amino acids, into the sameprotein. For example, in the presence of mutated O-tRNA's, e.g., aspecial frameshift suppressor tRNA's, with anticodon loops, e.g., with aCU(X)_(n), XXXAA sequence (where n=1), the four or more base codon isread as single amino acid. For example, see SEQ ID NOs.: 6, 12 fromPCT/US04/22061 for O-tRNA's that recognize a four base codon. In otherembodiments, the anticodon loops can decode, e.g., at least a four-basecodon, at least a five-base codon, or at least a six-base codon or more.Since there are 256 possible four-base codons, multiple unnatural aminoacids can be encoded in the same cell using a four or more base codon.See, Anderson et al., (2002) Exploring the Limits of Codon and AnticodonSize, Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding theGenetic Code: Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNA's. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol. Biol., 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in invention, which canreduce missense readthrough and frameshift suppression at other unwantedsites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology, 20:177-182. See, also, Wu, Y., etal., (2002) J. Am. Chem. Soc. 124:14626-14630. Other relevantpublications are listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274.A 3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., Tae et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the present invention can take advantageof this property to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate aselected amino acid, e.g., an unnatural amino acid, in a desiredpolypeptide. In a translational bypassing system, a large sequence isinserted into a gene but is not translated into protein. The sequencecontains a structure that serves as a cue to induce the ribosome to hopover the sequence and resume translation downstream of the insertion.

Selected and Unnatural Amino Acids

As used herein, a selected amino acid refers to any desired naturallyoccurring amino acid or unnatural amino acid. A naturally occurringamino acid includes any one of the twenty genetically encodedalpha-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine. In one embodiment, the selected amino acidis incorporated into a growing polypeptide chain with high fidelity,e.g., at greater than about 70% efficiency for given selector codon, atgreater than 75% efficiency for a given selector codon, at greater thanabout 80% efficiency for a given selector codon, at greater than about85% efficiency for a given selector codon, at greater than about 90%efficiency for a given selector codon, at greater than about 95%efficiency for a given selector codon, or at greater than about 99% ormore efficiency for a given selector codon.

As used herein, an unnatural amino acid refers to any amino acid,modified amino acid, or amino acid analogue other than selenocysteineand/or pyrrolysine and the following twenty genetically encodedalpha-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine. The generic structure of an alpha-aminoacid is illustrated by Formula I:

An unnatural amino acid is typically any structure having Formula Iwherein the R group is any substituent other than one used in the twentynatural amino acids. See, e.g., Biochemistry by L. Stryer, 3^(rd) ed.1988, Freeman and Company, New York, for structures of the twentynatural amino acids. Note that, the unnatural amino acids of the presentinvention can be naturally occurring compounds other than the twentyalpha-amino acids above.

Because the unnatural amino acids of the present invention typicallydiffer from the natural amino acids only in the structure of the sidechain, the unnatural amino acids form amide bonds with other aminoacids, including but not limited to, natural or unnatural, in the samemanner in which they are formed in naturally occurring proteins.However, the unnatural amino acids have side chain groups thatdistinguish them from the natural amino acids. For example, R in FormulaI may comprise an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-,hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol,seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine,amine, and the like, or any combination thereof. Other non-naturallyoccurring amino acids include, but are not limited to, amino acidscomprising a photoactivatable cross-linker, spin-labeled amino acids,fluorescent amino acids, metal binding amino acids, metal-containingamino acids, radioactive amino acids, amino acids with novel functionalgroups, amino acids that covalently or noncovalently interact with othermolecules, photocaged and/or photoisomerizable amino acids, amino acidscomprising biotin or a biotin analogue, glycosylated amino acids such asa sugar substituted serine, other carbohydrate modified amino acids,keto-containing amino acids, amino acids comprising polyethylene glycolor polyether, heavy atom substituted amino acids, chemically cleavableand/or photocleavable amino acids, amino acids with an elongated sidechains as compared to natural amino acids, including but not limited to,polyethers or long chain hydrocarbons, including but not limited to,greater than about 5 or greater than about 10 carbons, carbon-linkedsugar-containing amino acids, redox-active amino acids, amino thioacidcontaining amino acids, and amino acids comprising one or more toxicmoiety. See, also, U.S. Patent Application Publications 2003/0082575 and2003/0108885, which are incorporated by reference herein. Unnaturalamino acids may have a photoactivatable cross-linker that is used, e.g.,to link a protein to a solid support. Unnatural amino acids may have asaccharide moiety attached to the amino acid side chain.

In addition to unnatural amino acids that contain novel side chains,unnatural amino acids also optionally comprise modified backbonestructures, e.g., as illustrated by the structures of Formula II andIII:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids may comprise substitutions in the aminoor carboxyl group as illustrated by Formulas II and III. Unnatural aminoacids of this type include, but are not limited to, α-hydroxy acids,α-thioacids, α-aminothiocarboxylates, e.g., with side chainscorresponding to the common twenty natural amino acids or unnatural sidechains. In addition, substitutions at the α-carbon optionally include L,D, or α-α-disubstituted amino acids such as D-glutamate, D-alanine,D-methyl-O-tyrosine, aminobutyric acid, and the like. Other structuralalternatives include cyclic amino acids, such as proline analogues aswell as 3, 4, 6, 7, 8, and 9 membered ring proline analogues, β and γamino acids such as substituted β-alanine and γ-amino butyric acid.

Many unnatural amino acids are based on natural amino acids, such astyrosine, glutamine, phenylalanine, and the like. Tyrosine analogsinclude para-substituted tyrosines, ortho-substituted tyrosines, andmeta substituted tyrosines, wherein the substituted tyrosine comprises aketo group (including but not limited to, an acetyl group), a benzoylgroup, an amino group, a hydrazine, an hydroxyamine, a thiol group, acarboxy group, an isopropyl group, a methyl group, a C₆-C₂₀ straightchain or branched hydrocarbon, a saturated or unsaturated hydrocarbon,an O-methyl group, a polyether group, a nitro group, or the like. Inaddition, multiply substituted aryl rings are also contemplated.Glutamine analogs include, but are not limited to, α-hydroxyderivatives, γ-substituted derivatives, cyclic derivatives, and amidesubstituted glutamine derivatives. Example phenylalanine analogsinclude, but are not limited to, para-substituted phenylalanines,ortho-substituted phenyalanines, and meta-substituted phenylalanines,wherein the substituent comprises a hydroxy group, a methoxy group, amethyl group, an allyl group, an aldehyde, an azido, an iodo, a bromo, aketo group (including but not limited to, an acetyl group), or the like.Specific examples of unnatural amino acids include, but are not limitedto, a p-acetyl-L-phenylalanine, a p-propargyl-phenylalanine,O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine,a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine,an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike. Examples of structures of a variety of unnatural amino acids areprovided in, for example, WO 2002/085923 entitled “In vivo incorporationof unnatural amino acids,” which is incorporated by reference herein.See also Kiick et al., (2002) Incorporation of azides into recombinantproteins for chemoselective modification by the Staudinger ligation,PNAS 99:19-24, which is incorporated by reference herein, for additionalmethionine analogs.

A non-natural amino acid incorporated into a polypeptide at the aminoterminus can be composed of an R group that is any substituent otherthan one used in the twenty natural amino acids and a 2^(nd) reactivegroup different from the NH₂ group normally present in α-amino acids(see Formula I). A similar non-natural amino acid can be incorporated atthe carboxyl terminus with a 2^(nd) reactive group different from theCOOH group normally present in α-amino acids (see Formula I).

The unnatural amino acids of the invention may be selected or designedto provide additional characteristics unavailable in the twenty naturalamino acids. For example, unnatural amino acid may be optionallydesigned or selected to modify the biological properties of a protein,e.g., into which they are incorporated. For example, the followingproperties may be optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, solubility, stability,e.g., thermal, hydrolytic, oxidative, resistance to enzymaticdegradation, and the like, facility of purification and processing,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic activity, redox potential,half-life, ability to react with other molecules, e.g., covalently ornoncovalently, and the like.

The structures of a variety of unnatural amino acids are provided in,for example, FIGS. 16, 17, 18, 19, 26, and 29 of WO 2002/085923 entitled“In vivo incorporation of unnatural amino acids,” which is incorporatedby reference herein. The examples are not meant to be limiting in anyway of amino acids that may be attached to a tRNA of the presentinvention.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic or non-eukaryoticcell, or in vitro. Thus, in certain embodiments, the post-translationalmodification is through the unnatural amino acid. An unnatural aminoacid in a polypeptide may be used to attach another molecule to thepolypeptide, including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, or any combinationof the above or any other desirable compound or substance, comprising asecond reactive group to at least one unnatural amino acid comprising afirst reactive group utilizing chemistry methodology that is known toone of ordinary skill in the art to be suitable for the particularreactive groups.

For example, the post-translational modification can be through anucleophilic-electrophilic reaction. Most reactions currently used forthe selective modification of proteins involve covalent bond formationbetween nucleophilic and electrophilic reaction partners, including butnot limited to the reaction of α-haloketones with histidine or cysteineside chains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein. In proteinsof the invention, other more selective reactions can be used such as thereaction of an unnatural keto-amino acid with hydrazides or aminooxycompounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) J.Am. Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science,276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al.,(2002) J. Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl.Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci.,100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin,et al., (2003) Science, 301:964-7, all of which are incorporated byreference herein. This allows the selective labeling of virtually anyprotein with a host of reagents including fluorophores, crosslinkingagents, saccharide derivatives and cytotoxic molecules. See also, U.S.Pat. No. 6,927,042 entitled “Glycoprotein synthesis,” which isincorporated by reference herein. Post-translational modifications,including but not limited to, through an azido amino acid, can also madethrough the Staudinger ligation (including but not limited to, withtriarylphosphine reagents). See, e.g., Kiick et al., (2002)Incorporation of azides into recombinant proteins for chemoselectivemodification by the Staudinger ligation, PNAS 99:19-24.

Chemical Synthesis of Unnatural Amino Acids

Many unnatural amino acids are commercially available, e.g., fromSigma-Aldrich (St. Louis, Mo., USA), Novabiochem (a division of EMDBiosciences, Darmstadt, Germany), or Peptech (Burlington, Mass., USA).Those that are not commercially available are optionally synthesized asprovided herein or using standard methods known to those of ordinaryskill in the art. For organic synthesis techniques, see, e.g., OrganicChemistry by Fessendon and Fessendon, (1982, Second Edition, WillardGrant Press, Boston Mass.); Advanced Organic Chemistry by March (ThirdEdition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistryby Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press,New York). Additional publications describing the synthesis of unnaturalamino acids include, e.g., WO 2002/085923 entitled “In vivoincorporation of Unnatural Amino Acids;” Matsoukas et al., (1995) J.Med. Chem., 38, 4660-4669; King, F. E. & Kidd, D. A. A. (1949) A NewSynthesis of Glutamine and of γ-Dipeptides of Glutamic Acid fromPhthylated Intermediates. J. Chem. Soc., 3315-3319; Friedman, O. M. &Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as ModelSubstrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752;Craig, J. C. et al. (1988) Absolute Configuration of the Enantiomers of7-Chloro-4 [[4-(diethylamino)-1-methylbutyl]amino]quinoline(Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. &Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur.J. Med. Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989)Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. &Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates fromL-Asparagine. Application to the Total Synthesis of (+)-Apovincaminethrough Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem. 50:1239-1246; Barton et al., (1987) Synthesis of Novelalpha-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis ofL- and D-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid andAppropriate Unsaturated Derivatives. Tetrahedron 43:4297-4308; and,Subasinghe et al., (1992) Quisqualic acid analogues: synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activityat a novel quisqualate-sensitized site. J. Med. Chem. 35:4602-7. Seealso, U.S. Patent Publication No. US 2004/0198637 entitled “ProteinArrays,” which is incorporated by reference.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a cell is one issue that is typicallyconsidered when designing and selecting unnatural amino acids, e.g., forincorporation into a protein. For example, the high charge density ofα-amino acids suggests that these compounds are unlikely to be cellpermeable. Natural amino acids are taken up into the cell via acollection of protein-based transport systems. A rapid screen can bedone which assesses which unnatural amino acids, if any, are taken up bycells. See, e.g., the toxicity assays in, e.g., U.S. Patent PublicationNo. US 2004/0198637 entitled “Protein Arrays” which is incorporated byreference herein, and Liu, D. R. & Schultz, P. G. (1999) Progress towardthe evolution of an organism with an expanded genetic code. PNAS UnitedStates 96:4780-4785. Although uptake is easily analyzed with variousassays, an alternative to designing unnatural amino acids that areamenable to cellular uptake pathways is to provide biosynthetic pathwaysto create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, including butnot limited to, in a cell, the invention provides such methods. Forexample, biosynthetic pathways for unnatural amino acids are optionallygenerated in host cell by adding new enzymes or modifying existing hostcell pathways. Additional new enzymes are optionally naturally occurringenzymes or artificially evolved enzymes. For example, the biosynthesisof p-aminophenylalanine (as presented in an example in WO 2002/085923entitled “In vivo incorporation of unnatural amino acids”) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a cell by transformingthe cell with a plasmid comprising the genes. The genes, when expressedin the cell, provide an enzymatic pathway to synthesize the desiredcompound. Examples of the types of enzymes that are optionally added areprovided in the examples below. Additional enzymes sequences are found,for example, in Genbank. Artificially evolved enzymes are alsooptionally added into a cell in the same manner. In this manner, thecellular machinery and resources of a cell are manipulated to produceunnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, e.g., as developed by Maxygen, Inc.(available on the World Wide Web at maxygen.com), is optionally used todevelop novel enzymes and pathways. See, e.g., Stemmer (1994), Rapidevolution of a protein in vitro by DNA shuffling, Nature 370(4):389-391;and, Stemmer, (1994), DNA shuffling by random fragmentation andreassembly: In vitro recombination for molecular evolution, Proc. Natl.Acad. Sci. USA., 91:10747-10751. Similarly DesignPath™, developed byGenencor (available on the World Wide Web at genencor.com) is optionallyused for metabolic pathway engineering, e.g., to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to, those identified through functionalgenomics, and molecular evolution and design. Diversa Corporation(available on the World Wide Web at diversa.com) also providestechnology for rapidly screening libraries of genes and gene pathways,including but not limited to, to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the present invention is produced in aconcentration sufficient for efficient protein biosynthesis, e.g., anatural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a cell is transformed with a plasmid comprisingthe genes used to produce enzymes desired for a specific pathway and anunnatural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the unnatural amino acid forboth ribosomal protein synthesis and cell growth.

Nucleic Acid and Polypeptide Sequence and Variants

As described above and below, the invention provides for nucleic acidpolynucleotide sequences and polypeptide amino acid sequences, e.g.,tRNA's and RSs, and, e.g., compositions and methods comprising saidsequences. Examples of said sequences, e.g., tRNA's and RSs aredisclosed herein. However, one of skill in the art will appreciate thatthe invention is not limited to those sequences disclosed herein, e.g.,the Examples. One of skill will appreciate that the invention alsoprovides many related and unrelated sequences with the functionsdescribed herein, e.g., encoding an O-tRNA or an O-RS.

The invention provides polypeptides (O-RSs) and polynucleotides, e.g.,O-tRNA, polynucleotides that encode O-RSs or portions thereof,oligonucleotides used to isolate aminoacyl-tRNA synthetase clones, etc.Polynucleotides of the present invention include those that encodeproteins or polypeptides of interest of the present invention with oneor more selector codon. In addition, polynucleotides of the presentinvention include, e.g., a polynucleotide comprising a nucleotidesequence as set forth in SEQ ID NO.: 4; a polynucleotide that iscomplementary to or that encodes a polynucleotide sequence thereof, or aconservative variation thereof. A polynucleotide of the presentinvention also includes a polynucleotide that encodes a polypeptide ofthe present invention. Similarly, a nucleic acid that hybridizes to apolynucleotide indicated above under highly stringent conditions oversubstantially the entire length of the nucleic acid is a polynucleotideof the present invention. In one embodiment, a composition includes apolypeptide of the present invention and an excipient (e.g., buffer,water, pharmaceutically acceptable excipient, etc.). In addition,polypeptides of the present invention include, e.g., a polypeptidecomprising a amino acid sequence as set forth in SEQ ID NO.: 5; apolypeptide that is complementary to or that encodes a polypeptidesequence thereof, or a conservative variation thereof. The inventionalso provides an antibody or antisera specifically immunoreactive with apolypeptide of the present invention.

In certain embodiments, a vector (e.g., a plasmid, a cosmid, a phage, abacterium, a virus, a naked polynucleotide, a conjugated polynucleotide,etc.) comprises a polynucleotide of the present invention. In oneembodiment, the vector is an expression vector. In another embodiment,the expression vector includes a promoter operably linked to one or moreof the polynucleotides of the present invention. In another embodiment,a cell comprises a vector that includes a polynucleotide of the presentinvention.

One of skill will also appreciate that many variants of the disclosedsequences are included in the invention. For example, conservativevariations of the disclosed sequences that yield a functionallyidentical sequence are included in the invention. Variants of thenucleic acid polynucleotide sequences, wherein the variants hybridize toat least one disclosed sequence, are considered to be included in theinvention. Unique subsequences of the sequences disclosed herein, asdetermined by, e.g., standard sequence comparison techniques, are alsoincluded in the invention.

Conservative Variations

Owing to the degeneracy of the genetic code, “silent substitutions”(i.e., substitutions in a nucleic acid sequence which do not result inan alteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Similarly,“conservative amino acid substitutions,” in one or a few amino acids inan amino acid sequence are substituted with different amino acids withhighly similar properties, are also readily identified as being highlysimilar to a disclosed construct. Such conservative variations of eachdisclosed sequence are a feature of the present invention.

“Conservative variations” of a particular nucleic acid sequence refersto those nucleic acids which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences. One of ordinaryskill in the art will recognize that individual substitutions, deletionsor additions which alter, add or delete a single amino acid or a smallpercentage of amino acids in an encoded sequence are “conservativelymodified variations” or “conservatively modified variants” where thealterations result in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. Thus, “conservative variations” of a listed polypeptidesequence of the present invention include substitutions of a smallpercentage, typically less than 5%, more typically less than 4%, 2% or1%, of the amino acids of the polypeptide sequence, with aconservatively selected amino acid of the same conservative substitutiongroup. The addition of sequences which do not alter the encoded activityof a nucleic acid molecule, such as the addition of a non-functionalsequence, is a conservative variation of the basic nucleic acid.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3)Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (5),Threonine (T); and 8) Cysteine (C), Methionine (M)(see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)

Nucleic Acid Hybridization

Comparative hybridization can be used to identify nucleic acids of thepresent invention, such as SEQ ID NO.: 4, including conservativevariations of nucleic acids of the present invention, and thiscomparative hybridization method is a preferred method of distinguishingnucleic acids of the present invention. In addition, target nucleicacids which hybridize to the nucleic acids represented by SEQ ID NO: 4under high, ultra-high, and/or ultra-ultra high stringency conditionsare a feature of the present invention. Examples of such nucleic acidsinclude those with one or a few silent or conservative nucleic acidsubstitutions as compared to a given nucleic acid sequence.

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least ½ as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at least ½ as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 5×-10× as high as that observed for hybridization toany of the unmatched target nucleic acids.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. The phrase “stringent hybridizationconditions” refers to conditions of low ionic strength and hightemperature as is known in the art. Typically, under stringentconditions a probe will hybridize to its target subsequence in a complexmixture of nucleic acid (including but not limited to, total cellular orlibrary DNA or RNA) but does not hybridize to other sequences in thecomplex mixture. An extensive guide to the hybridization of nucleicacids is found in Tijssen (1993) Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Acid Probes part Ichapter 2, “Overview of principles of hybridization and the strategy ofnucleic acid probe assays,” (Elsevier, N.Y.), as well as in Ausubel, etal., Current Protocols in Molecular Biology (1995). Hames and Higgins(1995) Gene Probes 1 IRL Press at Oxford University Press, Oxford,England, (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes2 IRL Press at Oxford University Press, Oxford, England (Hames andHiggins 2) provide details on the synthesis, labeling, detection andquantification of DNA and RNA, including oligonucleotides. Generally,stringent conditions are selected to be about 5-10° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength pH. The T_(m) is the temperature (under defined ionicstrength, pH, and nucleic concentration) at which 50% of the probescomplementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions maybe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (including but not limited to, 10 to 50 nucleotides) and atleast about 60° C. for long probes (including but not limited to,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide. Forselective or specific hybridization, a positive signal may be at leasttwo times background, optionally 10 times background hybridization.Exemplary stringent hybridization conditions can be as following: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. Suchwashes can be performed for 5, 15, 30, 60, 120, or more minutes.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see, Sambrook, et al., Molecular Cloning, ALaboratory Manual (3rd ed. 2001) for a description of SSC buffer). Oftenthe high stringency wash is preceded by a low stringency wash to removebackground probe signal. An example low stringency wash is 2×SSC at 40°C. for 15 minutes. In general, a signal to noise ratio of 5× (or higher)than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and Northern hybridizationsare sequence dependent, and are different under different environmentalparameters. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen (1993), supra. and in Hames and Higgins, 1 and 2.Stringent hybridization and wash conditions can easily be determinedempirically for any test nucleic acid. For example, in determininghighly stringent hybridization and wash conditions, the hybridizationand wash conditions are gradually increased (e.g., by increasingtemperature, decreasing salt concentration, increasing detergentconcentration and/or increasing the concentration of organic solventssuch as formalin in the hybridization or wash), until a selected set ofcriteria are met. For example, the hybridization and wash conditions aregradually increased until a probe binds to a perfectly matchedcomplementary target with a signal to noise ratio that is at least 5× ashigh as that observed for hybridization of the probe to an unmatchedtarget.

“Very stringent” conditions are selected to be equal to the thermalmelting point (T_(m)) for a particular probe. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. For the purposesof the present invention, generally, “highly stringent” hybridizationand wash conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH.

“Ultra high-stringency” hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched targetnucleic acids. A target nucleic acid which hybridizes to a probe undersuch conditions, with a signal to noise ratio of at least ½ that of theperfectly matched complementary target nucleic acid is said to bind tothe probe under ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids. A target nucleic acid which hybridizesto a probe under such conditions, with a signal to noise ratio of atleast ½ that of the perfectly matched complementary target nucleic acidis said to bind to the probe under ultra-ultra-high stringencyconditions.

Nucleic acids which do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Unique Subsequences

In one aspect, the invention provides a nucleic acid that comprises aunique subsequence in a nucleic acid selected from the sequences ofO-tRNA's and O-RSs disclosed herein. The unique subsequence is unique ascompared to a nucleic acid corresponding to any known O-tRNA or O-RSnucleic acid sequence. Alignment can be performed using, e.g., BLAST setto default parameters. Any unique subsequence is useful, e.g., as aprobe to identify the nucleic acids of the present invention.

Similarly, the invention includes a polypeptide which comprises a uniquesubsequence in a polypeptide selected from the sequences of O-RSsdisclosed herein. Here, the unique subsequence is unique as compared toa polypeptide corresponding to any of known polypeptide sequence.

The invention also provides for target nucleic acids which hybridizesunder stringent conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from thesequences of O-RSs wherein the unique subsequence is unique as comparedto a polypeptide corresponding to any of the control polypeptides (e.g.,parental sequences from which synthetases of the present invention werederived, e.g., by mutation). Unique sequences are determined as notedabove.

Sequence Comparison, Identity, and Homology

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, or designated region, as measured using one of the sequencecomparison algorithms described below (or other algorithms available topersons of ordinary skill in the art) or by manual alignment and visualinspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding an O-tRNA or O-RS, or theamino acid sequence of an O—RS) refers to two or more sequences orsubsequences that have at least about 60%, about 80%, about 90-95%,about 98%, about 99% or more nucleotide or amino acid residue identity,when compared and aligned for maximum correspondence over a comparisonwindow, or designated region, as measured using a sequence comparisonalgorithm (or other algorithms available to persons of ordinary skill inthe art) or by manual alignment and visual inspection. Such“substantially identical” sequences are typically considered to be“homologous,” without reference to actual ancestry. “Substantialidentity” may exist over a region of the sequences that is at leastabout 50 residues in length, a region of at least about 100 residues, ora region of at least about 150 residues, or over the full length of thetwo sequences to be compared.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Methods of alignment of sequences for comparison are known to those ofordinary skill in the art. Optimal alignment of sequences for comparisoncan be conducted, e.g., by the local homology algorithm of Smith &Waterman, Adv. Appl. Math. 2:482c (1981), by the homology alignmentalgorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by thesearch for similarity method of Pearson & Lipman, Proc. Nat'l. Acad.Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see e.g., Ausubelet al., Current Protocols in Molecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm andBLAST 2.0 algorithms, which are described in Altschul et al., (1997)Nuc. Acids Res. 25:3389-3402, and Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Informationavailable at the World Wide Web at ncbi.nlm.nih.gov. This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always>0) and N (penalty score formismatching residues; always<0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1992) Proc. Natl.Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10,M=5, N=−4, and a comparison of both strands. The BLAST algorithm istypically performed with the “low complexity” filter turned off.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidmay be considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, less than about 0.01, or less thanabout 0.001.

Mutagenesis and Other Molecular Biology Techniques

Polynucleotide and polypeptides of the present invention and used in theinvention can be manipulated using molecular biological techniques. Anucleotide sequence may be conveniently modified by site-directedmutagenesis in accordance with conventional methods. Alternatively, thenucleotide sequence may be prepared by chemical synthesis, including butnot limited to, by using an oligonucleotide synthesizer, whereinoligonucleotides are designed based on the amino acid sequence of thedesired polypeptide, and preferably selecting those codons that arefavored in the host cell in which the recombinant polypeptide will beproduced. For example, several small oligonucleotides coding forportions of the desired polypeptide may be synthesized and assembled byPCR, ligation or ligation chain reaction. See, e.g., Barany, et al.,Proc. Natl. Acad. Sci. 88: 189-193 (1991); U.S. Pat. No. 6,521,427 whichare incorporated by reference herein.

This invention utilizes routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, e.g., thegeneration of genes or polynucleotides that include selector codons forproduction of proteins that include selected amino acids (e.g.,unnatural amino acids), orthogonal tRNA's, orthogonal synthetases, andpairs thereof.

Various types of mutagenesis are used in the invention for a variety ofpurposes, including but not limited to, to produce novel synthetases ortRNAs, to mutate tRNA molecules, to produce libraries of tRNAs, tomutate RS molecules, to produce libraries of synthetases, to produceselector codons, to insert selector codons that encode a selected aminoacid in a protein or polypeptide of interest. They include but are notlimited to site-directed, random point mutagenesis, homologousrecombination, DNA shuffling or other recursive mutagenesis methods,chimeric construction, mutagenesis using uracil containing templates,oligonucleotide-directed mutagenesis, phosphorothioate-modified DNAmutagenesis, mutagenesis using gapped duplex DNA or the like, or anycombination thereof. Additional suitable methods include point mismatchrepair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like. Mutagenesis, including but not limited to,involving chimeric constructs, is also included in the presentinvention. In one embodiment, mutagenesis can be guided by knowninformation of the naturally occurring molecule or altered or mutatednaturally occurring molecule, including but not limited to, sequence,sequence comparisons, physical properties, secondary, tertiary, orquaternary structure, crystal structure or the like.

The texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling et al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Zoller & Smith, Oligonucleotide-directed mutagenesis usingM13-derived vectors: an efficient and general procedure for theproduction of point mutations in any DNA fragment, Nucleic Acids Res.10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directedmutagenesis of DNA fragments cloned into M13 vectors, Methods inEnzymol. 100:468-500 (1983); Zoller & Smith, Oligonucleotide-directedmutagenesis: a simple method using two oligonucleotide primers and asingle-stranded DNA template, Methods in Enzymol. 154:329-350 (1987);Taylor et al., The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764(1985); Taylor et al., The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res. 13: 8765-8785 (1985); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Sayers et al., 5′-3′ Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Different base/base mismatches arecorrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E. coli, Cell 38:879-887 (1984); Carter etal., Improved oligonucleotide site-directed mutagenesis using M13vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Improvedoligonucleotide-directed mutagenesis using M13 vectors, Methods inEnzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use ofoligonucleotides to generate large deletions, Nucl. Acids Res. 14: 5115(1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakmar and Khorana, Total synthesis and expression of a gene forthe alpha-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Grundströmet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001); W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Oligonucleotides, e.g., for use in mutagenesis of the present invention,e.g., mutating libraries of tRNAs or synthetases, or altering tRNAs orRSs, are typically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers,Tetrahedron Letts. 22(20):1859-1862, (1981) e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al., Nucleic AcidsRes., 12:6159-6168 (1984).

In addition, essentially any nucleic acid can be custom or standardordered from any of a variety of commercial sources, such as The MidlandCertified Reagent Company (mcrc@oligos.com), The Great American GeneCompany (www.genco.com), ExpressGen Inc. (www.expressgen.com), OperonTechnologies Inc. (Alameda, Calif.) and many others.

The invention also relates to eukaryotic host cells, non-eukaryotic hostcells, and organisms for the in vivo incorporation of an unnatural aminoacid via orthogonal tRNAIRS pairs. Host cells are genetically engineered(including but not limited to, transformed, transduced or transfected)with the polynucleotides of the present invention or constructs whichinclude a polynucleotide of the present invention, including but notlimited to, a vector of the present invention, which can be, forexample, a cloning vector or an expression vector. For example, thecoding regions for the orthogonal tRNA, the orthogonal tRNA synthetase,and the protein to be derivatized are operably linked to gene expressioncontrol elements that are functional in the desired host cell. Thevector can be, for example, in the form of a plasmid, a cosmid, a phage,a bacterium, a virus, a naked polynucleotide, or a conjugatedpolynucleotide. The vectors are introduced into cells and/ormicroorganisms by standard methods including electroporation (Fromm etal., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), infection by viralvectors, high velocity ballistic penetration by small particles with thenucleic acid either within the matrix of small beads or particles, or onthe surface (Klein et al., Nature 327, 70-73 (1987)), and/or the like.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in the invention. Theseinclude: fusion of the recipient cells with bacterial protoplastscontaining the DNA, electroporation, projectile bombardment, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, kits arecommercially available for the purification of plasmids from bacteria,(see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™ from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect cells or incorporated into related vectorsto infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both (including but not limited to,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and/orintegration in prokaryotes, eukaryotes, or both. See, Gillam & Smith,Gene 8:81 (1979); Roberts, et al., Nature, 328:731 (1987); Schneider,E., et al., Protein Expr. Purif. 6(1)10-14 (1995); Ausubel, Sambrook,Berger (all supra). A catalogue of bacteria and bacteriophages usefulfor cloning is provided, e.g., by the ATCC, e.g., The ATCC Catalogue ofBacteria and Bacteriophage (1992) Gherna et al. (eds) published by theATCC. Additional basic procedures for sequencing, cloning and otheraspects of molecular biology and underlying theoretical considerationsare also found in Watson et al. (1992) Recombinant DNA Second EditionScientific American Books, NY. In addition, essentially any nucleic acid(and virtually any labeled nucleic acid, whether standard ornon-standard) can be custom or standard ordered from any of a variety ofcommercial sources, such as the Midland Certified Reagent Company(Midland, Tex. available on the World Wide Web at mcrc.com), The GreatAmerican Gene Company (Ramona, Calif. available on the World Wide Web atgenco.com), ExpressGen Inc. (Chicago, Ill. available on the World WideWeb at expressgen.com), Operon Technologies Inc. (Alameda, Calif.) andmany others.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, e.g. for cell isolation and culture (e.g., for subsequentnucleic acid isolation) include Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein; Payne et al. (1992) Plant Cell and TissueCulture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York) and Atlas and Parks (eds.) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla.

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers a wide variety of advantages including but not limited tohigh yields of mutant proteins, technical ease, the potential to studythe mutant proteins in cells or possibly in living organisms and the useof these mutant proteins in therapeutic treatments and diagnostic uses.The ability to include unnatural amino acids with various sizes,acidities, nucleophilicities, hydrophobicities, and other propertiesinto proteins can greatly expand our ability to rationally andsystematically manipulate the structures of proteins, both to probeprotein function and create new proteins or organisms with novelproperties.

Proteins and Polypeptides of Interest

The incorporation of an unnatural amino acid can be done for a varietyof purposes, including but not limited to, tailoring changes in proteinstructure and/or function, changing size, acidity, nucleophilicity,hydrogen bonding, hydrophobicity, accessibility of protease targetsites, targeting to a moiety (including but not limited to, for aprotein array), adding a biologically active molecule, attaching apolymer, attaching a radionuclide, modulating serum half-life,modulating tissue penetration (e.g. tumors), modulating activetransport, modulating tissue, cell or organ specificity or distribution,modulating immunogenicity, modulating protease resistance, etc. Proteinsthat include an unnatural amino acid can have enhanced or even entirelynew catalytic or biophysical properties. For example, the followingproperties are optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic ability, half-life (including but not limited to, serumhalf-life), ability to react with other molecules, including but notlimited to, covalently or noncovalently, and the like. The compositionsincluding proteins that include at least one unnatural amino acid areuseful for, including but not limited to, novel therapeutics,diagnostics, catalytic enzymes, industrial enzymes, binding proteins(including but not limited to, antibodies), and including but notlimited to, the study of protein structure and function. See, e.g.,Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structureand Function, Current Opinion in Chemical Biology, 4:645-652.

A protein may have at least one, including but not limited to, at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, or at least ten or moreunnatural amino acids. The unnatural amino acids can be the same ordifferent, including but not limited to, there can be 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 or more different sites in the protein that comprise 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnatural amino acids. Aprotein may have at least one, but fewer than all, of a particular aminoacid present in the protein is substituted with the unnatural aminoacid. For a given protein with more than one unnatural amino acids, theunnatural amino acids can be identical or different (including but notlimited to, the protein can include two or more different types ofunnatural amino acids, or can include two of the same unnatural aminoacid). For a given protein with more than two unnatural amino acids, theunnatural amino acids can be the same, different or a combination of amultiple unnatural amino acid of the same kind with at least onedifferent unnatural amino acid.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in eukaryotic cells, proteins or polypeptides willtypically include eukaryotic post-translational modifications. Incertain embodiments, a protein includes at least one unnatural aminoacid and at least one post-translational modification that is made invivo by a eukaryotic cell, where the post-translational modification isnot made by a prokaryotic cell. For example, the post-translationmodification includes, including but not limited to, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, glycolipid-linkage modification, glycosylation, and thelike. In yet another aspect, the post-translation modification includesproteolytic processing of precursors (including but not limited to,calcitonin precursor, calcitonin gene-related peptide precursor,preproparathyroid hormone, preproinsulin, proinsulin,prepro-opiomelanocortin, pro-opiomelanocortin and the like), assemblyinto a multisubunit protein or macromolecular assembly, translation toanother site in the cell (including but not limited to, to organelles,such as the endoplasmic reticulum, the Golgi apparatus, the nucleus,lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., orthrough the secretory pathway). In certain embodiments, the proteincomprises a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, or the like.

Methods of producing a protein in a cell with a selected amino acid at aspecified position are also a feature of the present invention. Forexample, a method includes growing, in an appropriate medium, the cell,where the cell comprises a nucleic acid that comprises at least oneselector codon and encodes a protein; and, providing the selected aminoacid; where the cell further comprises: an orthogonal tRNA (O-tRNA) thatfunctions in the cell and recognizes the selector codon; and, anorthogonal aminoacyl-tRNA synthetase (O-RS) that preferentiallyaminoacylates the O-tRNA with the selected amino acid. Typically, theO-tRNA comprises suppression activity in presence of a cognatesynthetase in response to a selector codon. A protein produced by thismethod is also a feature of the present invention.

The compositions of the present invention and compositions made by themethods of the present invention optionally are in a cell. TheO-tRNA/O-RS pairs or individual components of the present invention canthen be used in a host system's translation machinery, which results ina selected amino acid, e.g., unnatural amino acid, being incorporatedinto a protein. Patent application U.S. Ser. Nos. 10/825,867, entitled“Expanding the Eukaryotic Genetic Code;” and 10/126,927, entitled “INVIVO INCORPORATION OF UNNATURAL AMINO ACIDS”, describe this process andare incorporated herein by reference. For example, when an O-tRNA/O-RSpair is introduced into a host, e.g., Escherichia coli, the pair leadsto the in vivo incorporation of selected amino acid, such as anunnatural amino acid, e.g., a synthetic amino acid, such as derivativeof a leucine amino acid, which can be exogenously added to the growthmedium, into a protein, in response to a selector codon. Optionally, thecompositions of the present invention can be in an in vitro translationsystem, or in an in vivo system(s).

Any protein (or portion thereof) that includes a selected amino acid,e.g., an unnatural amino acid, (and any corresponding coding nucleicacid, e.g., which includes one or more selector codons) can be producedusing the compositions and methods herein. Any polypeptide is suitablefor incorporation of one or more selected amino acids. No attempt ismade to identify the hundreds of thousands of known proteins, any ofwhich can be modified to include one or more unnatural amino acid, e.g.,by tailoring any available mutation methods to include one or moreappropriate selector codon in a relevant translation system. Commonsequence repositories for known proteins include GenBank EMBL, DDBJ andthe NCBI. Other repositories can easily be identified by searching theinterne.

Typically, the proteins are, e.g., at least 60%, at least 70%, at least75%, at least 80%, at least 90%, at least 95%, or at least 99% or moreidentical to any available protein (e.g., a therapeutic protein, adiagnostic protein, an industrial enzyme, or portion thereof, and thelike), and they comprise one or more selected amino acid. Examples oftherapeutic, diagnostic, and other proteins that can be modified tocomprise one or more selected amino acid, e.g., an unnatural amino acid,can be found, but not limited to, those in U.S. Ser. No. 10/825,867entitled “Expanding the Eukaryotic Genetic Code;” and, U.S. patentapplication Ser. No. 10/126,927, entitled “IN VIVO INCORPORATION OFUNNATURAL AMINO ACIDS.”

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the presentinvention is encoded by a nucleic acid. Typically, the nucleic acidcomprises at least one selector codon, at least two selector codons, atleast three selector codons, at least four selector codons, at leastfive selector codons, at least six selector codons, at least sevenselector codons, at least eight selector codons, at least nine selectorcodons, ten or more selector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods well-known to one of skill in the art and described hereinunder “Mutagenesis and Other Molecular Biology Techniques” to include,e.g., one or more selector codon for the incorporation of a selectedamino acid, e.g., an unnatural amino acid. For example, a nucleic acidfor a protein of interest is mutagenized to include one or more selectorcodon, providing for the insertion of the one or more selected aminoacids, e.g., unnatural amino acid. The invention includes any suchvariant, e.g., mutant, versions of any protein, e.g., including at leastone selected amino acid. Similarly, the invention also includescorresponding nucleic acids, i.e., any nucleic acid with one or moreselector codon that encodes one or more selected amino acid.

To make a protein that includes a selected amino acid, one can use hostcells and organisms that are adapted for the in vivo incorporation ofthe selected amino acid via orthogonal tRNA/RS pairs. Host cells aregenetically engineered (e.g., transformed, transduced or transfected)with one or more vectors that express the orthogonal tRNA, theorthogonal tRNA synthetase, and a vector that encodes the protein to bederivatized. Each of these components can be on the same vector, or eachcan be on a separate vector, two components can be on one vector and thethird component on a second vector. The vector can be, for example, inthe form of a plasmid, a cosmid, a phage, a bacterium, a virus, a nakedpolynucleotide, or a conjugated polynucleotide.

Alternate Systems

Several strategies have been employed to introduce unnatural amino acidsinto proteins in non-recombinant host cells, mutagenized host cells, orin cell-free systems. Derivatization of amino acids with reactiveside-chains such as Lys, Cys and Tyr resulted in the conversion oflysine to N²-acetyl-lysine. Chemical synthesis also provides astraightforward method to incorporate unnatural amino acids. With therecent development of enzymatic ligation and native chemical ligation ofpeptide fragments, it is possible to make larger proteins. See, e.g., P.E. Dawson and S. B. H. Kent, Annu. Rev. Biochem, 69:923 (2000). Chemicalpeptide ligation and native chemical ligation are described in U.S. Pat.No. 6,184,344, U.S. Patent Publication No. 2004/0138412, U.S. PatentPublication No. 2003/0208046, WO 02/098902, and WO 03/042235, which areincorporated by reference herein. A general in vitro biosynthetic methodin which a suppressor tRNA chemically acylated with the desiredunnatural amino acid is added to an in vitro extract capable ofsupporting protein biosynthesis, has been used to site-specificallyincorporate over 100 unnatural amino acids into a variety of proteins ofvirtually any size. See, e.g., V. W. Cornish, D. Mendel and P. G.Schultz, Angew. Chem. Int. Ed. Engl., 1995, 34:621 (1995); C. J. Noren,S. J. Anthony-Cahill, M. C. Griffith, P. G. Schultz, A general methodfor site-specific incorporation of unnatural amino acids into proteins,Science 244:182-188 (1989); and, J. D. Bain, C. G. Glabe, T. A. Dix, A.R. Chamberlin, E. S. Diala, Biosynthetic site-specific incorporation ofa non-natural amino acid into a polypeptide, J. Am. Chem. Soc.111:8013-8014 (1989). A broad range of functional groups has beenintroduced into proteins for studies of protein stability, proteinfolding, enzyme mechanism, and signal transduction.

An in vivo method, termed selective pressure incorporation, wasdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophic strain, inwhich the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been incorporated in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Karnbrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind,L. Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionineanalogs with alkene or alkyne functionalities have also beenincorporated efficiently, allowing for additional modification ofproteins by chemical means. See, e.g., J. C. van Hest and D. A. Tirrell,FEBS Lett., 428:68 (1998); J. C. van Hest, K. L. Kiick and D. A.Tirrell, J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick and D. A.Tirrell, Tetrahedron, 56:9487 (2000); U.S. Pat. No. 6,586,207; U.S.Patent Publication 2002/0042097, which are incorporated by referenceherein.

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. For example, replacement of Ala²⁹⁴ by Gly inEscherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the sizeof substrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS. Nishimura, J. Biol. Chem., 275:40324 (2000).

Another strategy to incorporate unnatural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAValwith Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys (—SH group of Cys is replaced with —CH3 inAbu), the mutant ValRS also incorporates Abu into proteins when thismutant Escherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing novel amino acids. Forexample, see the following publications and references cited within,which are as follows: Crick, F. H. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides.XXXVI. The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am. Chem,88(24):5914-5919 (1966); Kaiser, E. T. Synthetic approaches tobiologically active peptides and proteins including enyzmes, Acc ChemRes, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptidesegment coupling catalyzed by the semisynthetic enzyme thiosubtilisin, JAm Chem Soc, 109:3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256(5054):221-225 (1992);Chaiken, I. M. Semisynthetic peptides and proteins, CRC Crit RevBiochem, 11(3):255-301 (1981); Offord, R. E. Protein engineering bychemical means? Protein Eng., 1(3):151-157 (1987); and, Jackson, D. Y.,Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J. A. A DesignedPeptide Ligase for Total Synthesis of Ribonuclease A with UnnaturalCatalytic Residues, Science, 266(5183):243 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation ofa hybrid sequence-specific single-stranded deoxyribonuclease, Science,238(4832):1401-1403 (1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E.The chemical modification of enzymatic specificity, Annu Rev Biochem,54:565-595 (1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation ofenyzme active sites, Science, 226(4674):505-511 (1984); Neet, K. E.,Nanci A, Koshland, D. E. Properties of thiol-subtilisin, J Biol. Chem,243(24):6392-6401 (1968); Polgar, L. et M. L. Bender. A new enzymecontaining a synthetically formed active site. Thiol-subtilisin. J. AmChem Soc, 88:3153-3154 (1966); and, Pollack, S. J., Nakayama, G.Schultz, P. G. Introduction of nucleophiles and spectroscopic probesinto antibody combining sites, Science, 242(4881):1038-1040 (1988).

Defining Polypeptides by Immunoreactivity

Because the polypeptides of the present invention provide a variety ofnew polypeptide sequences (e.g., comprising selected amino acids (e.g.,unnatural amino acids) in the case of proteins synthesized in thetranslation systems herein, or, e.g., in the case of the novelsynthetases, novel sequences of standard amino acids), the polypeptidesalso provide new structural features which can be recognized, e.g., inimmunological assays. The generation of antisera, which specificallybind the polypeptides of the present invention, as well as thepolypeptides which are bound by such antisera, are a feature of thepresent invention. The term “antibody,” as used herein, includes, but isnot limited to a polypeptide substantially encoded by an immunoglobulingene or immunoglobulin genes, or fragments thereof which specificallybind and recognize an analyte (antigen). Examples include polyclonal,monoclonal, chimeric, and single chain antibodies, and the like.Fragments of immunoglobulins, including Fab fragments and fragmentsproduced by an expression library, including phage display, are alsoincluded in the term “antibody” as used herein. See, e.g., Paul,Fundamental Immunology, 4th Ed., 1999, Raven Press, New York, forantibody structure and terminology.

For example, the invention includes RSs and proteins made utilizingtRNAs and/or RSs of the present invention that specifically bind to orthat are specifically immunoreactive with an antibody or antiseragenerated against an immunogen comprising an amino acid sequence. Toeliminate cross-reactivity with other homologues, the antibody orantisera is subtracted with available protein, such as the wild-typepolypeptide, e.g., the “control” polypeptides. Where the wild-typeprotein corresponds to a nucleic acid, a polypeptide encoded by thenucleic acid is generated and used for antibody/antisera subtractionpurposes.

In one typical format, the immunoassay uses a polyclonal antiserum whichwas raised against one or more polypeptide or a substantial subsequencethereof (i.e., at least about 30% of the full length sequence provided).The set of potential polypeptide immunogens derived from the protein arecollectively referred to below as “the immunogenic polypeptides.” Theresulting antisera is optionally selected to have low cross-reactivityagainst the control synthetase homologues and any such cross-reactivityis removed, e.g., by immunoabsorbtion, with one or more of the controlhomologues, prior to use of the polyclonal antiserum in the immunoassay.

In order to produce antisera for use in an immunoassay, one or more ofthe immunogenic polypeptides is produced and purified as describedherein. For example, recombinant protein can be produced in arecombinant cell. An inbred strain of mice (used in this assay becauseresults are more reproducible due to the virtual genetic identity of themice) is immunized with the immunogenic protein(s) in combination with astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see, e.g., Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for astandard description of antibody generation, immunoassay formats andconditions that can be used to determine specific immunoreactivity.

Additional references and discussion of antibodies is also found hereinand can be applied here to defining polypeptides by immunoreactivity).Alternatively, one or more synthetic or recombinant polypeptide derivedfrom the sequences disclosed herein is conjugated to a carrier proteinand used as an immunogen.

Polyclonal sera are collected and titered against the immunogenicpolypeptide in an immunoassay, for example, a solid phase immunoassaywith one or more of the immunogenic proteins immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁶ or greater areselected, pooled and subtracted with the control synthetase polypeptidesto produce subtracted pooled titered polyclonal antisera.

The subtracted pooled titered polyclonal antisera are tested for crossreactivity against the control homologues in a comparative immunoassay.In this comparative assay, discriminatory binding conditions aredetermined for the subtracted titered polyclonal antisera which resultin at least about a 5-10 fold higher signal to noise ratio for bindingof the titered polyclonal antisera to the immunogenic protein ascompared to binding to the control synthetase homologues. That is, thestringency of the binding reaction is adjusted by the addition ofnon-specific competitors such as albumin or non-fat dry milk, and/or byadjusting salt conditions, temperature, and/or the like. These bindingconditions are used in subsequent assays for determining whether a testpolypeptide (a polypeptide being compared to the immunogenicpolypeptides and/or the control polypeptides) is specifically bound bythe pooled subtracted polyclonal antisera.

In another example, immunoassays in the competitive binding format areused for detection of a test polypeptide. For example, as noted,cross-reacting antibodies are removed from the pooled antisera mixtureby immunoabsorbtion with the control polypeptides. The immunogenicpolypeptide(s) are then immobilized to a solid support which is exposedto the subtracted pooled antisera. Test proteins are added to the assayto compete for binding to the pooled subtracted antisera. The ability ofthe test protein(s) to compete for binding to the pooled subtractedantisera as compared to the immobilized protein(s) is compared to theability of the immunogenic polypeptide(s) added to the assay to competefor binding (the immunogenic polypeptides compete effectively with theimmobilized immunogenic polypeptides for binding to the pooledantisera). The percent cross-reactivity for the test proteins iscalculated, using standard calculations.

In a parallel assay, the ability of the control proteins to compete forbinding to the pooled subtracted antisera is optionally determined ascompared to the ability of the immunogenic polypeptide(s) to compete forbinding to the antisera. Again, the percent cross-reactivity for thecontrol polypeptides is calculated, using standard calculations. Wherethe percent cross-reactivity is at least 5-10× as high for the testpolypeptides as compared to the control polypeptides and or where thebinding of the test polypeptides is approximately in the range of thebinding of the immunogenic polypeptides, the test polypeptides are saidto specifically bind the pooled subtracted antisera.

In general, the immunoabsorbed and pooled antisera can be used in acompetitive binding immunoassay as described herein to compare any testpolypeptide to the immunogenic and/or control polypeptide(s). In orderto make this comparison, the immunogenic, test and control polypeptidesare each assayed at a wide range of concentrations and the amount ofeach polypeptide required to inhibit 50% of the binding of thesubtracted antisera to, e.g., an immobilized control, test orimmunogenic protein is determined using standard techniques. If theamount of the test polypeptide required for binding in the competitiveassay is less than twice the amount of the immunogenic polypeptide thatis required, then the test polypeptide is said to specifically bind toan antibody generated to the immunogenic protein, provided the amount isat least about 5-10× as high as for the control polypeptide.

As an additional determination of specificity, the pooled antisera isoptionally fully immunosorbed with the immunogenic polypeptide(s)(rather than the control polypeptides) until little or no binding of theresulting immunogenic polypeptide subtracted pooled antisera to theimmunogenic polypeptide(s) used in the immunosorbtion is detectable.This fully immunosorbed antisera is then tested for reactivity with thetest polypeptide. If little or no reactivity is observed (i.e., no morethan 2× the signal to noise ratio observed for binding of the fullyimmunosorbed antisera to the immunogenic polypeptide), then the testpolypeptide is specifically bound by the antisera elicited by theimmunogenic protein.

Additional details on proteins, antibodies, antisera, etc. can be foundin U.S. Ser. No. 10/825,867 entitled “Expanding the Eukaryotic GeneticCode;” WO 2002/085923, entitled “IN VIVO INCORPORATION OF UNNATURALAMINO ACIDS;” U.S. Pat. No. 6,927,042 entitled “Glycoprotein synthesis”;and U.S. Patent Publication No. US 2004/0198637 entitled “ProteinArrays,” which is incorporated by reference.

Kits

Kits are also a feature of the present invention. For example, a kit forproducing a protein that comprises at least one selected amino acid,e.g., an unnatural amino acid, in a cell is provided, where the kitincludes a container containing a polynucleotide sequence encoding anO-tRNA, and/or an O-tRNA, and/or a polynucleotide sequence encoding anO—RS, and/or an O-RS. In one embodiment, the kit further includes atleast selected amino acid. In another embodiment, the kit includes anaminoacylated tRNA of the invention. In another embodiment, the kitfurther comprises instructional materials for producing the protein.

An additional example is a kit for producing a protein that comprises atleast one selected amino acid, e.g., an unnatural amino acid, in acell-free translation system, where the kit includes a containercontaining a polynucleotide sequence encoding an O-tRNA, and/or anO-tRNA, and/or a polynucleotide sequence encoding an O-RS, and/or anO-RS. In one embodiment, the kit further includes a selected amino acid.In another embodiment, the kit includes an aminoacylated tRNA of theinvention. In another embodiment, the kit further comprisesinstructional materials for producing the protein.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. One of skill will recognize a variety of non-criticalparameters that may be altered without departing from the scope of theclaimed invention.

Example 1 Aminoacyl-tRNA Synthetase Selection Against Para-AcetylPhenylalanine

Two DNA libraries were screened for aminoacyl-tRNA synthetases againstpara-acetyl phenylalanine, a non-naturally encoded amino acid. Theselibraries consisted of six mutations in the tyrosyl tRNA synthetase genefrom Methanococcous janneschii in the pBK plasmid.

The selection procedure was preformed which consisted of fivealternating rounds of selection, three positive, two negative. Thelibraries were combined in a 1:1 ratio and electroporated into thepositive selection cell line (GeneHog with positive selection plasmid,pREP) and plated on minimal media plates (GMML) with appropriateantibiotics and the non-naturally encoded amino acid para-acetylphenylalanine (pAF). The plates were incubated at 37° C. for about 40hours at which point the cells were harvested by scraping. The DNA wasextracted using a Qiagen Mini-Prep procedure, and then was agarose gelpurified to isolate the library plasmid DNA.

This DNA was then electroporated into the negative selection cell line(GeneHog with negative selection plasmid pBAD derivative). Thesetransformants were plated on LB plates with appropriate antibioticwithout the non-naturally encoded amino acid (pAF). After about 17 hoursthese cells were harvested by scraping and the plasmid DNA was purifiedusing the Qiagen Mini-Prep procedure and agarose gel purification.

The subsequent rounds of selection were done utilizing the same methodof electroporation, plating, harvesting, and DNA purification. In thelast (fifth) round of selection, serial dilutions were made of thetransformed positive selection cells which were plated on minimal mediaplates. Individual colonies were then picked and grown in a 96 wellblock overnight. This block was then replica plated on minimal mediaplates with varying concentrations of choloramphenicol (the positiveselection antibiotic) with and without unnatural amino acid pAF. Afterabout 40 hours of growth at 37° C., the plates were visually compared todetermine which colonies grew on the highest chloroamphenicolconcentration but did not grow or grew poorly in the absence of thenon-naturally encoded amino acid pAF. The colonies which met thesecriteria were grown overnight. The DNA was isolated from the cultures byMini-Prep and agarose gel purification and were sequenced.

From this selection for pAF, 13 clones were found to have unique aminoacid sequences and were subjected to further characterization todetermine the fidelity and processivity of the pAF-tRNA synthetase.

To characterize these synthetases, small scale amber suppressions wereperformed to show that the non-naturally encoded amino acid pAF wasincorporated into a polypeptide, and the results were visualized bySDS-PAGE. A single colony was picked and grown overnight in LB broth,which was then used to inoculate 50 mL of LB. The cells were grown to anOD of 0.3-0.4, at which point 1.5 mL aliquots were taken aspre-induction points and the culture was split into two flasks. 1 mM pAFwas added to one split and both were grown for 30 minutes. Following the30 minute growth, both cultures (+/−pAF) were induced with 0.2%L-Arabinose and grown 4.5 hours and the OD₆₀₀ was recorded. 1.5 mLaliquots were then taken of the +/−pAF flasks for SDS-PAGE analysis.

The 1.5 mL aliquots (Preinduction, +pAF, −pAF) were centrifuged at10,000×g for 10 minutes to pellet the cells. The cells were thensuspended in proportional Bacterial Protein Extraction Reagent (BPER,Pierce) amounts relative to their OD₆₀₀ at the time of harvest. DNase Iwas added to the lysed cells and incubated at 4° C. for 20 minutes. Thesamples were then combined with a reducing agent and loading dye and runon a 4-12% Bis-TRIS gel in MES buffer for 30 minutes. The gel was washedin DI H₂O twice for 10 minutes and stained with coommassie blue dye. The+/−pAF bands were compared for the fidelity of the pAF-tRNA RS to resultin incorporation of pAF, and the +pAF band was compared to thepreviously selected pAF-tRNA RS.

To check for the processivity of the RSs the same procedure wasperformed with a plasmid containing C—H6 S4am myoglobin (S4am-Myo). TheS4am Myo was then purified by IMAC and sent for protein sequencing todetermine the amount of pAF incorporation.

Of the pAF-tRNA RSs identified from this selection one synthetase (E9)was found to incorporate pAF efficiently, with greater than 95%efficiency of incorporation of pAF into S4am-Myo. The incorporation wasdetermined by amino acid sequencing while the processivity was shown bycomparing protein bands on a SDS-PAGE gels. The nucleotide sequence forE9 is shown in SEQ ID NO: 4, and the amino acid sequence of E9 is shownin SEQ ID NO: 5.

An additional mutant with similar activity to E9 was identified, and hasthe amino acid sequence shown in SEQ ID NO: 17.

Example 2 tRNA Mutagenesis

Three mutants were generated of tRNA J17. The DNA sequence of wild-typeJ17 is shown as SEQ ID NO: 8 and in U.S. Patent Publication Nos.2003/0108885 as SEQ ID NO: 1 and US 2003/0082575 as SEQ ID NO: 1 (U.S.patent application Ser. Nos. 10/126,931 and 10/126,927, respectively),both of which are incorporated by reference herein. J17 tRNA has aU51:G63 wobble pair in the TΨC stem as shown in FIG. 1.

Three J17 mutants (F12, F13, and F14) were generated to produceWatson-Crick base pairs at positions 51 and 63 of the TΨC stem.Mutagenesis was performed by overlapping PCR, and the final constructswere cloned into EcoRI and NdeI sites in a pET19 plasmid comprising thepolynucleotide sequence encoding the aminoacyl tRNA synthetase E9 (SEQID NO: 4) and the polynucleotide sequence encoding human growth hormone(hGH) with an amber codon substitution (SEQ ID NO: 16). The expressionof hGH was under the control of the T7 promoter.

Two fragments were generated for overlapping PCR. The first fragment wasobtained by primer extension. The sequence of the forward primer used togenerate each of the three mutants was:GTAACGCTGAATTCCCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAAT CCGCATGGCGC(FTam11; SEQ ID NO: 9).

To generate the F12 mutant (51C:63G), the following reverse primer wasused: GATCTGCAGTGGTCCGGCGGGCCGGATTTGAACCGGCGCCATGCGGATTTAGAGTCCGCCGTTCTGC (FTam12; SEQ ID NO: 10).

To generate the F13 mutant (51U:63A), the following reverse primer wasused: GATCTGCAGTGGTCCGGCGGGCTGGATTTGAACCAGCGCCATGCGGATTTAGAGTCCGCCGTTCTGC (FTam13; SEQ ID NO: 11).

To generate the F14 mutant (51A:63U), the following reverse primer wasused: GATCTGCAGTGGTCCGGCGGGCAGGATTTGAACCTGCGCCATGCGGATTTAGAGTCCGCCGTTCTGC (FTam14; SEQ ID NO: 12).

To generate the second fragment, plasmid pET19 J17 E9 hGH comprising thepolynucleotide sequence for J17 tRNA (SEQ ID NO: 8), the polynucleotidesequence encoding the tRNA synthetase E9 (SEQ ID NO: 4) and thepolynucleotide sequence encoding human growth hormone with an ambercodon substitution (SEQ ID NO: 16) was used as a template foramplification with the following set of primers:CGCCGGACCACTGCAGATCCTTAGCGAAAGCTAAGGATTTTTTTTAAGC (forward primer;FTam15; SEQ ID NO: 13) and CAAATTCGTCCATATGGGATTCC (FTam 16; SEQ ID NO:14). The forward primer was used to extend the sequence from the 3′ endof tRNA to, the Nde I site of the plasmid. The resulting product was gelpurified.

The final step of overlapping PCR involved forward primerGTAACGCTGAATTCCCGGCG (FTam17, SEQ ID NO: 15), reverse primer FTam16 (SEQID NO: 14), the first fragment and the second fragment. The assembledproducts were digested with EcoR I and Nde I and ligated into theplasmid pET19 J17 E9 hGH digested with EcoR I and Nde I. The sequence ofeach construct was confirmed by sequencing, and the DNA sequences foreach of the J17 mutant tRNAs are shown as SEQ ID NO: 1 (F12), SEQ ID NO:2 (F13), and SEQ ID NO: 3 (F14). The tRNAs were named after theircorresponding reverse primers.

Protein Expression

Plasmids encoding the tRNAs (J17, F12, F13 or F14) were each transformedinto E. coli strain 1 and strain 2 bacterial host cells by chemicalmeans and plated onto LB agar plates with 50 ug/ml carbenicillin. Theplates were incubated at 37° C. overnight. For each tRNA, a singlecolony was picked to start an overnight culture at 37° C. in 1 ml 2×YTwith 50 ug/ml carbenicillin. This 1 ml culture was used to inoculate two10 ml 2×YT cultures with 50 ug/ml carbenicillin at 37° C. One 10 mlculture was supplemented with 4 mM para-acetylphenylalanine. AtOD₆₀₀=0.7, the hGH expression was induced with 0.4 mM IPTG. Afterculturing the cells at 37° C. for 4 hours with 250 rpm, the cells wereharvested by centrifugation at 5000×g for 5 minutes. The cells werelysed with B-PER Reagent (Pierce, Rockford, Ill.) supplemented with 5ug/ml DNAse I. The total cell lysate was analyzed by 4-12% SDS PAGE.

FIG. 2 shows an analysis of E. coli strain 1 total cell lysates by SDSPAGE. Supression of a selector codon in human growth hormone wasperformed using J17 or J17 mutant (F12, F13, F14) tRNA and the aminoacyltRNA synthetase E9. Cells harboring J17 mutants grew slightly slowerthan cells harboring J17. No full length hGH product was observed bySDS-PAGE for the tRNA mutants in the absence of 4 mMpara-acetylphenylalanine. In the presence of 4 mMpara-acetylphenylalanine, full length product was produced with each ofthe tRNA mutants, demonstrating that these tRNA mutant-RS E9 pairs areorthogonal to E. coli machinery. Based on SDS-PAGE, the suppressed hGHyield of the J17 mutants was approximately 1.5˜2 fold higher than thatof J17 in E. coli strain 1.

One J17 mutant, F13, was further tested in E. coli strain 2 bacterialcell line for amber suppression as shown in FIG. 3. In E. coli strain 2,the expression as well as amber suppression yields were reduced relativeto that in E. coli strain 1. In the absence of para-acetylphenylalanine,no full length hGH product was observed by SDS-PAGE. In the presence 4mM para-acetylphenylalanine, full length hGH was observed for bothtRNAs. Based on SDS-PAGE, the suppressed hGH yield of F13 was aboutthree fold higher than that of J17.

A fermentation run comparing J17 and F13 was performed with a finalvolume of approximately 1.5 L. The plasmid encoding the J17 tRNA and theplasmid encoding F13 tRNA were each transformed into E. coli strain 1.The final cell density for each was approximately 190 g wet cells/1. ThehGH titer was 347 mg/L for the J17 clone and 542 mg/L for the F13 clone.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of thepresent invention. For example, all the techniques and apparatusdescribed above can be used in various combinations. All publications,patents, patent applications, and/or other documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication, patent,patent application, and/or other document were individually indicated tobe incorporated by reference for all purposes.

TABLE 1 SEQ ID NO: Label SEQUENCE 1 F12CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCA DNATGGCGCCGGTTCAAATCCGGCCCGCCGGACCA 2 F13CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCA DNATGGCGCTGGTTCAAATCCAGCCCGCCGGACCA 3 F14CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCA DNATGGCGCAGGTTCAAATCCTGCCCGCCGGACCA 4 E9 RSATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATC nucleicAGCGAGGAAGAGTTAAGAGAGGTTTTAAAAAAAGATGAAAAATC acidTGCTGTTATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATATTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAACATGGTCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGGATTCATTATGAGGGCGATGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTITGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATFTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATT AGAAAGAGATTATAA 5 E9 RSMDEFEMIKRNTSEIISEEELREVLKKDEKSAVIGFEPSGKIHLGHYLQIK AminoKMIDLQNAGFDIIIYLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGL AcidKAKYVYGSEHGLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGIHYEGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDL KNAVAEELIKILEPIRKRL 6HL(TAG)3 CCCAGGGTAGCCAAGCTCGGCCAACGGCGACGGACTCTAAATCCG tRNATTCTCGTAGGAGTTCGAGGGTTCGAATCCCTTCCCTGGGACCA DNA 7 HL(TGA)1GCGGGGGTTGCCGAGCCTGGCCAAAGGCGCCGGACTTCAAATCCG tRNAGTCCCGTAGGGGTTCCGGGGTTCAAATCCCCGCCCCCGCACCA DNA 8 J17CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCA M.TGGCGCTGGTTCAAATCCGGCCCGCCGGACCA jannaschii mtRNA_(CUA) ^(Tyr) DNA 9FTam11 GTAACGCTGAATTCCCGGCGGTAGTTCAGCAGGGCAGAACGGCGG primerACTCTAAATCCGCATGGCGC 10 FTam12GATCTGCAGTGGTCCGGCGGGCCGGATTTGAACCGGCGCCATGCG primerGATTTAGAGTCCGCCGTTCTGC 11 FTam13GATCTGCAGTGGTCCGGCGGGCTGGATTTGAACCAGCGCCATGCG primerGATTTAGAGTCCGCCGTTCTGC 12 FTam14GATCTGCAGTGGTCCGGCGGGCAGGATTTGAACCTGCGCCATGCG primerGATTTAGAGTCCGCCGTTCTGC 13 FTam15CGCCGGACCACTGCAGATCCTTAGCGAAAGCTAAGGATTTTTTTTA primer AGC 14 FTam16CAAATTCGTCCATATGGGATTCC primer 15 FTam17 GTAACGCTGAATTCCCGGCG primer 16hGH ATGGGCCACCACCACCACCACCACTTCCCAACCATTCCCTTATCCA (DNA)GGCTTTTTTGACAACGCTATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAGGAGTTTGAAGAAGCCTAGATCCCAAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACCCTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATCTCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTCCTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTGACAGCAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGT GGCTTCTAA 17 D286RMDEFEMIKRN TSEIISEEEL REVLKKDEKS AVIGFEPSGK mutant ofIHLGHYLQIK KMIDLQNAGF DIIIYLADLH AYLNQKGELD E9EIRKIGDYNK KVFEAMGLKA KYVYGSEHGL DKDYTLNVYRLALKTTLKRA RRSMELIARE DENPKVAEVI YPIMQVNGIHYEGVDVAVGG MEQRKIHMLA RELLPKKVVC IHNPVLTGLDGEGKMSSSKG NFIAVDDSPE EIRAKIKKAY CPAGVVEGNPIMEIAKYFLE YPLTIKRPEK FGGDLTVNSY EELESLFKNK ELHPMRLKNA VAEELIKILE PIRKRL

1. A cell-free composition comprising a nucleic acid sequence thatencodes the aminoacyl tRNA synthetase (RS) sequence of SEQ ID NO: 17,and the complementary polynucleotide sequence thereof.
 2. Thecomposition of claim 1, wherein said RS preferentially aminoacylates atRNA.
 3. The composition of claim 2, wherein said tRNA is aminoacylatedwith a non-naturally encoded amino acid.
 4. The composition of claim 3,wherein said non-naturally encoded amino acid ispara-acetylphenylalanine.
 5. The composition of claim 2, wherein saidtRNA is enzymatically aminoacylated by the RS.
 6. The composition ofclaim 1, further comprising a tRNA, wherein the tRNA is aminoacylatedwith an amino acid by the RS.
 7. The composition of claim 6, whereinsaid amino acid is a non-naturally encoded amino acid.
 8. Thecomposition of claim 7, wherein said non-naturally encoded amino acid ispara-acetylphenylalanine.
 9. The composition of claim 6, wherein thetRNA is derived from an archael tRNA.
 10. The composition of claim 6,wherein the tRNA is derived from M. janneschii.
 11. The composition ofclaim 1, further comprising a translation system.
 12. The composition ofclaim 11, wherein said translation system is a cell lysate.
 13. Thecomposition of claim 11, wherein said translation system is areconstituted system.
 14. The composition of claim 11, wherein saidtranslation system is a cellular translation system.